Methods and compositions for detecting bacterial nucleic acid and diagnosing bacterial vaginosis

ABSTRACT

Disclosed are methods for diagnosing Bacterial Vaginosis in a subject comprising performing an assay for the detection of any one or more of Lactobacillus sp., Gardneralla vaginalis, and Eggerthella sp. in a subject sample. Also disclosed are methods and compositions for detecting Lactobacillus sp., Gardneralla vaginalis, and/or Eggerthella nucleic acid in a sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage entry of International PatentApplication No. PCT/US2016/022628, filed Mar. 16, 2016, and claimsbenefit of priority under 35 U.S.C § 119(e) to provisional applicationNo. 62/133,881, filed Mar. 16, 2015, 62/168,405, filed May 29, 2015, and62/168,688, filed May 29, 2015; the entire contents of each areincorporated herein by reference

REFERENCE TO SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII Copy, created on Aug. 8, 2017, isnamed “DIA-0002-05-UT_ST25.txt” and is 31 KB in size.

BACKGROUND

According to the National Health and Nutrition Examination Survey,nearly a third of women between the age of 14 and 49 have bacterialvaginosis (BV). (See Allsworth and Peipert, Obstetrics and Gynecology109:114-120, 2007). BV is the most common cause of vaginal discharge anda reason many women seek medical attention. It is also associated withpreterm birth, low birth weight, pelvic inflammatory disease, anincrease in STD infections, including HIV, and a greater risk of passingHIV on to sex partners. See Srinivasan and Fredricks, InterdisciplinaryPerspectives on Infectious Diseases, Vol. 2008, Article ID 750479, 22pages, 2008). Women with bacterial vaginosis may have symptoms includinga malodorous vaginal discharge or irritation, however, as many as halfof the women with diagnosable BV have no clear symptoms (see Srivinvasanand Fredricks, supra).

No single etiologic agent is known to be the cause of BV. Mostresearchers and the CDC consider bacterial vaginosis to be the result ofa disruption to the normal bacterial flora of the vagina. Unlike commoninfections, this dysbiosis is not the result of an individual bacterialspecies. See CDC Factsheet, 2014 (BV-Fact-Sheet-March-2014.pdf, from CDCwebsite). A dysbiosis is a disruption of the normal microbiota within abody environment such as the vagina. See Nibali et al., Journal of OralMicrobiology 6:22962, 2014.

BV is diagnosed in the clinic using the Amsel Criteria and in thelaboratory using the Nugent Scoring System. The later relies on countingbacterial morphotypes with the aid of the Gram stain. In this way, theNugent Score is a visual assessment of dysbiosis—it scores the badbacteria against the good. See Nugent et al., Journal of ClinicalMicrobiology 29:297-301, 1991. The Amsel Criteria evaluates a sample forthe presence of clue cells, pH, color and odor which are key symptomsassociated with BV. See Amsel et al., Am. J. Med. 74:14-22, 1983. A wetmount of the sample is examined with a microscope to detect clue cellswhich are human epithelial cells covered with bacteria thought topredominately consist of G. vaginalis.

Molecular tests generally target multiple organisms which have strongcorrelations with bacterial vaginosis. Which organisms are targetedvaries from test to test. In nearly all cases, high abundance anaerobicbacteria are targeted such as Atopobium, Gardnerella, and Megasphaeraspecies.

The only FDA approved test for BV (BD Affirm VPIII 2010), was found tohave a sensitivity of 67.6% and a specificity of 76.4% in a study byCartwright et al. (Journal of Clinical Microbiology 51:3694-3699, 2013).For the purpose diagnosing BV, the Affirm product detects G. vaginalisas its sole indicator. The product package insert indicates the Affirmproduct is 95.1% sensitive and 83.3% specific when compared to a scoredgram stain method.

Cartwright et al., supra, used a multiplex assay for the detection ofAtopobium vaginae, BVAB-2 and Megasphaera-1 for the diagnosis of BV.They measured the performance of this assay against a combination ofNugent and Amsel results in a population of 323 women (93%African-American, 7% white non-Hispanic). They reported this test was96.9% sensitive and 92.6% specific when compared to the combination ofNugent and Amsel scores. They did not report the results of this assayrelative to the Nugent Score alone.

SUMMARY

In one aspect, the present invention provides a method for determiningthe presence or absence of Bacterial Vaginosis (BV) in a subject. Insome embodiments, the method generally includes the following steps: (a)providing a sample from a subject suspected of having BV; (b) performingan assay for the detection of Lactobacillus sp., G. vaginalis, andEggerthella sp. in the sample; (c) for each of Lactobacillus sp., G.vaginalis, and Eggerthella sp., assigning a status of either positive ornegative based on the detection assay; and (d) determining the presenceor absence of BV in the subject based on a combination of the assignedLactobacillus sp. status, G. vaginalis status, and Eggerthella sp.status from step (c), where (i) a negative status for both G. vaginalisand Eggerthella sp. indicates the absence of BV in the subject, (ii) apositive status for both G. vaginalis and Eggerthella sp. indicates thepresence of BV in the subject, (iii) if the status of Lactobacillus sp.is positive, then a negative status for at least one of G. vaginalis andEggerthella indicates the absence of BV in the subject, and (iv) if thestatus of Lactobacillus sp. is negative, then a positive status for atleast one of G. vaginalis and Eggerthella indicates the presence of BVin the subject. In other embodiments, the method generally includes thefollowing steps: (a) providing a sample from a subject suspected ofhaving BV; (b) performing an assay for the detection of Lactobacillussp. and G. vaginalis in the sample; (c) for each of Lactobacillus sp.and G. vaginalis, assigning a status of either positive or negativebased on the detection assay; and (d) determining the presence orabsence of BV in the subject based on a combination of the assignedLactobacillus sp. status and G. vaginalis status from step (c), where(i) a negative status for G. vaginalis indicates the absence of BV inthe subject, and (ii) if the status of G. vaginalis is positive, then apositive status for Lactobacillus sp. indicates the absence of BV in thesubject and a negative status for Lactobacillus sp. indicates thepresence of BV in the subject.

In some embodiments of a method for diagnosing BV as above, the assayfor detection of Lactobacillus sp., G. vaginalis, and Eggerthella sp.,or the assay for detection of Lactobacillus sp. and G. vaginalis, is anucleic-acid-based detection assay. In some embodiements theLactobacillus sp. is from the vaginal microbiome. In some embodimentsthe Eggerthella sp. is from the vaginal microbiome. Particularlysuitable nucleic-acid-based detection assays include amplification-basedassays such as, for example, an assay comprising an isothermalamplification reaction (e.g., a transcription-mediated amplification(TMA) reaction), which may be performed in real time. In certainembodiments, the nucleic-acid-based detection assay targets the 16S rRNAof Lactobacillus sp., G. vaginalis, and Eggerthella sp., or the 16S rRNAof Lactobacillus sp. and G. vaginalis. In particular variations, thenucleic-acid-based detection assay targets (i) a Lactobacillus sp. 16SrRNA region corresponding to a region of SEQ ID NO:1 from aboutnucleotide position 91 to about nucleotide position 265; (ii) a G.vaginalis 16S rRNA region corresponding to a region of SEQ ID NO:3 fromabout nucleotide position 964 to about nucleotide position 1036; and/or(iii) an Eggerthella sp. 16S rRNA region corresponding to a region ofSEQ ID NO:4 from about nucleotide position 165 to about nucleotideposition 259.

In certain embodiments of a method for diagnosing BV as above comprisinga nucleic-acid-based detection assay, the assay is anamplification-based assay including the following steps:

-   -   (1) contacting the sample with        -   first and second Lactobacillus-specific amplification            oligomers for amplifying a target region of a Lactobacillus            sp. target nucleic acid, where (i) the first            Lactobacillus-specific amplification oligomer comprises a            first Lactobacillus-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            residues 28-45 of SEQ ID NO:7 or residues 28-45 of SEQ ID            NO:8 and (ii) the second Lactobacillus-specific            amplification oligomer comprises a second            Lactobacillus-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            SEQ ID NO:6;        -   first and second G. vaginalis-specific amplification            oligomers for amplifying a target region of a G. vaginalis            target nucleic acid, where (i) the first G.            vaginalis-specific amplification oligomer comprises a            first G. vaginalis-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            residues 28-45 of SEQ ID NO:13 and (ii) the second G.            vaginalis-specific amplification oligomer comprises a            second G. vaginalis-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            SEQ ID NO:12; and        -   first and second Eggerthella-specific amplification            oligomers for amplifying a target region of an Eggerthella            sp. target nucleic acid, where (i) the first            Eggerthella-specific amplification oligomer comprises a            first Eggerthella-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            residues 28-51 of SEQ ID NO:17 and (ii) the second            Eggerthella-specific amplification oligomer comprises a            second Eggerthella-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            SEQ ID NO:16;    -   (2) performing an in vitro nucleic acid amplification reaction,        where any Lactobacillus sp., G. vaginalis, and Eggerthella sp.        target nucleic acid, if present in the sample, is used as a        template for generating one or more amplification products        corresponding to the Lactobacillus sp., G. vaginalis, and        Eggerthella sp. target regions; and    -   (3) detecting the presence or absence of the one or more        amplification products.

In other embodiments of a method for diagnosing BV comprising anucleic-acid-based detection assay, the assay is an amplification-basedassay including the following steps:

-   -   (1) contacting the sample with        -   first and second Lactobacillus-specific amplification            oligomers for amplifying a target region of a Lactobacillus            sp. target nucleic acid, where (i) the first            Lactobacillus-specific amplification oligomer comprises a            first Lactobacillus-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            residues 28-45 of SEQ ID NO:7 or residues 28-45 of SEQ ID            NO:8 and (ii) the second Lactobacillus-specific            amplification oligomer comprises a second            Lactobacillus-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            SEQ ID NO:6; and        -   first and second G. vaginalis-specific amplification            oligomers for amplifying a target region of a G. vaginalis            target nucleic acid, where (i) the first G.            vaginalis-specific amplification oligomer comprises a            first G. vaginalis-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            residues 28-45 of SEQ ID NO:13 and (ii) the second G.            vaginalis-specific amplification oligomer comprises a            second G. vaginalis-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            SEQ ID NO:12;    -   (2) performing an in vitro nucleic acid amplification reaction,        where any Lactobacillus sp. and G. vaginalis target nucleic        acid, if present in the sample, is used as a template for        generating one or more amplification products corresponding to        the Lactobacillus sp. and G. vaginalis target regions; and    -   (3) detecting the presence or absence of the one or more        amplification products.

In some variations of a method for diagnosing BV comprising anamplification-based detection assay as above, the firstLactobacillus-specific target-hybridizing sequence comprises thenucleotide sequence of residues 28-45 of SEQ ID NO:7 or residues 28-45of SEQ ID NO:8; the second Lactobacillus-specific target-hybridizingsequence comprises the nucleotide sequence of SEQ ID NO:6; the first G.vaginalis-specific target-hybridizing sequence comprises the nucleotidesequence of residues 28-45 of SEQ ID NO:13; the second G.vaginalis-specific target-hybridizing sequence comprises the nucleotidesequence of SEQ ID NO:12; the first Eggerthella-specifictarget-hybridizing sequence comprises the nucleotide sequence ofresidues 28-51 of SEQ ID NO:17; and/or the second Eggerthella-specifictarget-hybridizing sequence comprises the nucleotide sequence of SEQ IDNO:16. In some such embodiments, the first Lactobacillus-specifictarget-hybridizing sequence consists of the nucleotide sequence ofresidues 28-45 of SEQ ID NO:7 or residues 28-45 of SEQ ID NO:8; thesecond Lactobacillus-specific target-hybridizing sequence consists ofthe nucleotide sequence of SEQ ID NO:6; the first G. vaginalis-specifictarget-hybridizing sequence consists of the nucleotide sequence ofresidues 28-45 of SEQ ID NO:13; the second G. vaginalis-specifictarget-hybridizing sequence consists of the nucleotide sequence of SEQID NO:12; the first Eggerthella-specific target-hybridizing sequenceconsists of the nucleotide sequence of residues 28-51 of SEQ ID NO:17;and/or the second Eggerthella-specific target-hybridizing sequenceconsists of the nucleotide sequence of SEQ ID NO:16.

In some variations of a method for diagnosing BV comprising anamplification-based detection assay as above, the firstLactobacillus-specific target-hybridizing sequence substantiallycorresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO:7,and the sample is further contacted with a third Lactobacillus-specificamplification oligomer for amplifying the Lactobacillus sp. targetregion, where the third Lactobacillus-specific amplification oligomerincludes a third Lactobacillus-specific target-hybridizing sequencesubstantially corresponding to the nucleotide sequence of residues 28-45of SEQ ID NO:8. In some such embodiments, the thirdLactobacillus-specific target-hybridizing sequence comprises thenucleotide sequence of residues 28-45 of SEQ ID NO:8. In a particularvariation, the third Lactobacillus-specific target-hybridizing sequenceconsists of the nucleotide sequence of residues 28-45 of SEQ ID NO:8.

In some embodiments of a method for diagnosing BV comprising anamplification-based detection assay as above, at least one of the firstLactobacillus-specific amplification oligomer, the first G.vaginalis-specific amplification oligomer, and the firstEggerthella-specific amplification oligomer (or at least one of thefirst Lactobacillus-specific amplification oligomer and the first G.vaginalis-specific amplification oligomer) is a promoter primer orpromoter provider further comprising a promoter sequence located 5′ tothe respective target hybridizing sequence. In some such embodimentswhere the first Lactobacillus-specific target-hybridizing sequencesubstantially corresponds to the nucleotide sequence of residues 28-45of SEQ ID NO:7 and the sample is further contacted with a thirdLactobacillus-specific amplification oligomer for amplifying theLactobacillus sp. target region, the third Lactobacillus-specificamplification oligomer is a promoter primer or promoter provider furthercomprising a promoter sequence located 5′ to the thirdLactobacillus-specific target hybridizing sequence. A particularlysuitable promoter sequence is a T7 promoter sequence such as, forexample, a promoter sequence having the nucleotide sequence of residues1-27 of SEQ ID NO:7. In specific variations, the firstLactobacillus-specific amplification oligomer has the nucleotidesequence of SEQ ID NO:7; the first G. vaginalis-specific amplificationoligomer has the nucleotide sequence of SEQ ID NO:13; and/or the firstEggerthella-specific amplification oligomer has the nucleotide sequenceof SEQ ID NO:17. In some embodiments as above where the sample isfurther contacted with a third Lactobacillus-specific amplificationoligomer for amplifying the Lactobacillus sp. target region, the thirdLactobacillus-specific amplification oligomer has the nucleotidesequence of SEQ ID NO:8.

In certain embodiments of a method for diagnosing BV comprising anamplification-based detection assay as above, the method furtherincludes purifying the Lactobacillus, G. vaginalis, and Eggerthellatarget nucleic acids, or purifying the Lactobacillus and G. vaginalistarget nucleic acids, if present, from other components in the samplebefore step (2). In some such embodiments, the purifying step includescontacting the sample with at least one capture probe oligomercomprising a target-hybridizing sequence covalently attached to asequence or moiety that binds to an immobilized probe. For example, thesample may be contacted with a Lactobacillus-specific capture probeoligomer, a G. vaginalis-specific capture probe oligomer, and anEggerthella-specific capture probe oligomer, where each of theLactobacillus-specific, G. vaginalis-specific, and Eggerthella-specificcapture probe oligomers comprises a capture probe target-hybridizingsequence that specifically hybridizes to a target sequence within theLactobacillus, G. vaginalis, or Eggerthella target nucleic acid,respectively, and where each of the Lactobacillus-specific, G.vaginalis-specific, and Eggerthella-specific capture probetarget-hybridizing sequences is covalently attached to the sequence ormoiety that binds to the immobilized probe. In some embodiments directedto detection of Lactobacillus sp. and G. vaginalis, the sample may becontacted with a Lactobacillus-specific capture probe oligomer and a G.vaginalis-specific capture probe oligomer, where each of theLactobacillus-specific and G. vaginalis-specific capture probe oligomerscomprises a capture probe target-hybridizing sequence that specificallyhybridizes to a target sequence within the Lactobacillus or G. vaginalistarget nucleic acid, respectively, and where each of theLactobacillus-specific and G. vaginalis-specific capture probetarget-hybridizing sequences is covalently attached to the sequence ormoiety that binds to the immobilized probe. In specific variations, theLactobacillus-specific capture probe target-hybridizing sequencesubstantially corresponds to the nucleotide sequence of residues 1-12 ofSEQ ID NO:5, the G. vaginalis-specific capture probe target-hybridizingsequence substantially corresponds to the nucleotide sequence ofresidues 1-17 of SEQ ID NO:11, and/or the Eggerthella-specific captureprobe target hybridizing sequence substantially corresponds to thenucleotide sequence of residues 1-21 of SEQ ID NO:15. In someembodiments, the Lactobacillus-specific capture probe oligomer has thenucleotide sequence of SEQ ID NO:5, the G. vaginalis-specific captureprobe oligomer has the nucleotide sequence of SEQ ID NO:11, and/or theEggerthella-specific capture probe oligomer has the nucleotide sequenceof SEQ ID NO:15.

In some embodiments of a method for diagnosing BV comprising anamplification-based detection assay as above, the detecting step (3)includes (i) contacting the one or more amplification products with afirst Lactobacillus-specific detection probe that specificallyhybridizes to the Lactobacillus sp. target region, a first G.vaginalis-specific detection probe that specifically hybridizes to theG. vaginalis target region, and a first Eggerthella-specific detectionprobe that specifically hybridizes to the Eggerthella sp. target region,and (ii) detecting the presence or absence of any target-hybridizedLactobacillus-specific, G. vaginalis-specific, and/orEggerthella-specific detection probe. In some embodiments of a methodfor diagnosing BV comprising an amplification-based detection assay fordetection of Lactobacillus sp. and G. vaginalis as above, the detectingstep (3) includes (i) contacting the one or more amplification productswith a first Lactobacillus-specific detection probe that specificallyhybridizes to the Lactobacillus sp. target region and a first G.vaginalis-specific detection probe that specifically hybridizes to theG. vaginalis target region, and (ii) detecting the presence or absenceof any target-hybridized Lactobacillus-specific and/or G.vaginalis-specific detection probe. In some embodiments, the firstLactobacillus-specific detection probe comprises a target-hybridizingsequence substantially corresponding to SEQ ID NO:9, the first G.vaginalis-specific detection probe comprises a target-hybridizingsequence substantially corresponding to residues 1-19 of SEQ ID NO:14,and/or the first Eggerthella-specific detection probe comprises atarget-hybridizing sequence substantially corresponding to SEQ ID NO:18.In specific variations, the first Lactobacillus-specific detection probehas the nucleotide sequence of SEQ ID NO:9, the first G.vaginalis-specific detection probe has the nucleotide sequence of SEQ IDNO:14, and/or the first Eggerthella-specific detection probe has thenucleotide sequence of SEQ ID NO:18. In some embodiments as above, thedetecting step (3) further includes contacting the one or moreamplification products with a second Lactobacillus-specific detectionprobe that specifically hybridizes to the Lactobacillus sp. targetregion, where the second Lactobacillus-specific detection probecomprises a target-hybridizing sequence substantially corresponding toresidues 6-21 of SEQ ID NO:10; in some such embodiments, the secondLactobacillus-specific detection probe has the nucleotide sequence ofSEQ ID NO:10.

In certain embodiments of a method for diagnosing BV comprising anamplification-based detection assay and the use of a firstLactobacillus-specific detection probe, a first G. vaginalis-specificdetection probe, and a first Eggerthella-specific detection probe (orthe use of a first Lactobacillus-specific detection probe and a first G.vaginalis-specific detection probe) as above, each of said probescomprises a label. In some embodiments further including contacting theone or more amplification products with a second Lactobacillus-specificdetection probe, the second Lactobacillus-specific detection probecomprises a label. Particularly suitable labels include chemiluminescentand fluorescent labels.

In some embodiments of a method for diagnosing BV comprising anamplification-based detection assay and the use of labeled detectionprobes as above, the detecting step (3) occurs during the amplifyingstep (2). In some such variations, each detection probe comprises afluorescent label and a quencher. Particularly suitable detection probescomprising a fluorescent label and a quencher include a molecular torch,a molecular beacon, and a TaqMan detection probe.

In certain embodiments of a method for diagnosing BV comprising anamplification-based detection assay and the use of detection probes asabove, at least one of the first Lactobacillus-specific detection probe,the first G. vaginalis-specific detection probe, and the firstEggerthella-specific detection probe (or at least one of the firstLactobacillus-specific detection probe and the first G.vaginalis-specific detection probe) further comprises anon-target-hybridizing sequence. In some embodiments further includingcontacting the one or more amplification products with a secondLactobacillus-specific detection probe, the secondLactobacillus-specific detection probe further comprises anon-target-hybridizing sequence. In some such variations as above, anyone of (e.g., each of) the detection probes is a molecular torch or amolecular beacon.

In some embodiments of a method for diagnosing BV comprising anamplification-based detection assay as above, the amplification reactionat step (2) is an isothermal amplification reaction. In particularvariations, the isothermal amplification reaction is atranscription-mediated amplification (TMA) reaction. In certainembodiments, the isothermal amplification reaction is a real-timeamplification reaction.

In some embodiments of a method for diagnosing BV as above, the methodincludes the detection of no more than ten bacterial genera associatedwith BV. For example, in certain variations, the method includes thedetection of no more than five bacterial genera associated with BV. In aspecific variation, the method does not include detection of bacterialgenera associated with BV other than Lactobacillus, Gardnerella, andEggerthella. In another specific variation, the method does not includedetection of bacterial genera associated with BV other thanLactobacillus and Gardnerella.

In some embodiments of a method for diagnosing BV as above, if thepresence of BV is indicated in the subject, then the method furtherincludes administering a treatment regime for BV to the subject.

In some embodiments of a method for diagnosing BV as above, the methodis a method for monitoring BV in the subject and the subject isundergoing a treatment regime for BV prior to step (a). In some suchvariations, if the presence of BV is indicated in the subject, then themethod further includes either (i) administering the treatment regimefor BV to the subject or (ii) administering a different treatment regimefor BV to the subject.

In another aspect, the present invention provides a multiplex detectionmethod. In some embodiments, the multiplex detection method is fordetermining the presence or absence of each of each of Lactobacillussp., G. vaginalis, and Eggerthella sp. in a sample. The method generallyincludes the following steps:

-   -   (1) contacting a sample, the sample suspected of containing at        least one of Lactobacillus sp., G. vaginalis, and Eggerthella        sp., with        -   (a) first and second Lactobacillus-specific amplification            oligomers for amplifying a target region of a Lactobacillus            sp. target nucleic acid, where (i) the first            Lactobacillus-specific amplification oligomer comprises a            first Lactobacillus-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            residues 28-45 of SEQ ID NO:7 or residues 28-45 of SEQ ID            NO:8 and (ii) the second Lactobacillus-specific            amplification oligomer comprises a second            Lactobacillus-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            SEQ ID NO:6;        -   (b) first and second G. vaginalis-specific amplification            oligomers for amplifying a target region of a G. vaginalis            target nucleic acid, where (i) the first G.            vaginalis-specific amplification oligomer comprises a            first G. vaginalis-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            residues 28-45 of SEQ ID NO:13 and (ii) the second G.            vaginalis-specific amplification oligomer comprises a            second G. vaginalis-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            SEQ ID NO:12; and        -   (c) first and second Eggerthella-specific amplification            oligomers for amplifying a target region of an Eggerthella            sp. target nucleic acid, where (i) the first            Eggerthella-specific amplification oligomer comprises a            first Eggerthella-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            residues 28-51 of SEQ ID NO:17 and (ii) the second            Eggerthella-specific amplification oligomer comprises a            second Eggerthella-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            SEQ ID NO:16;    -   (2) performing an in vitro nucleic acid amplification reaction,        where any Lactobacillus sp., G. vaginalis, and Eggerthella sp.        target nucleic acid, if present in the sample, is used as a        template for generating one or more amplification products        corresponding to the Lactobacillus sp., G. vaginalis, and        Eggerthella sp. target regions; and    -   (3) detecting the presence or absence of the one or more        amplification products, thereby determining the presence or        absence of Lactobacillus sp., G. vaginalis, and Eggerthella sp.        in the sample.

In other embodiments, the multiplex detection method is for determiningthe presence or absence of each of each of Lactobacillus sp. and G.vaginalis in a sample. The method generally includes the followingsteps:

-   -   (1) contacting a sample, the sample suspected of containing at        least one of Lactobacillus sp. and G. vaginalis, with        -   (a) first and second Lactobacillus-specific amplification            oligomers for amplifying a target region of a Lactobacillus            sp. target nucleic acid, where (i) the first            Lactobacillus-specific amplification oligomer comprises a            first Lactobacillus-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            residues 28-45 of SEQ ID NO:7 or residues 28-45 of SEQ ID            NO:8 and (ii) the second Lactobacillus-specific            amplification oligomer comprises a second            Lactobacillus-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            SEQ ID NO:6; and        -   (b) first and second G. vaginalis-specific amplification            oligomers for amplifying a target region of a G. vaginalis            target nucleic acid, where (i) the first G.            vaginalis-specific amplification oligomer comprises a            first G. vaginalis-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            residues 28-45 of SEQ ID NO:13 and (ii) the second G.            vaginalis-specific amplification oligomer comprises a            second G. vaginalis-specific target-hybridizing sequence            substantially corresponding to the nucleotide sequence of            SEQ ID NO:12;    -   (2) performing an in vitro nucleic acid amplification reaction,        where any Lactobacillus sp. and G. vaginalis target nucleic        acid, if present in the sample, is used as a template for        generating one or more amplification products corresponding to        the Lactobacillus sp. and G. vaginalis target regions; and    -   (3) detecting the presence or absence of the one or more        amplification products, thereby determining the presence or        absence of Lactobacillus sp. and G. vaginalis in the sample.

In some variations of a multiplex method as above, the firstLactobacillus-specific target-hybridizing sequence comprises thenucleotide sequence of residues 28-45 of SEQ ID NO:7 or residues 28-45of SEQ ID NO:8; the second Lactobacillus-specific target-hybridizingsequence comprises the nucleotide sequence of SEQ ID NO:6; the first G.vaginalis-specific target-hybridizing sequence comprises the nucleotidesequence of residues 28-45 of SEQ ID NO:13; the second G.vaginalis-specific target-hybridizing sequence comprises the nucleotidesequence of SEQ ID NO:12; the first Eggerthella-specifictarget-hybridizing sequence comprises the nucleotide sequence ofresidues 28-51 of SEQ ID NO:17; and/or the second Eggerthella-specifictarget-hybridizing sequence comprises the nucleotide sequence of SEQ IDNO:16. In some such embodiments, the first Lactobacillus-specifictarget-hybridizing sequence consists of the nucleotide sequence ofresidues 28-45 of SEQ ID NO:7 or residues 28-45 of SEQ ID NO:8; thesecond Lactobacillus-specific target-hybridizing sequence consists ofthe nucleotide sequence of SEQ ID NO:6; the first G. vaginalis-specifictarget-hybridizing sequence consists of the nucleotide sequence ofresidues 28-45 of SEQ ID NO:13; the second G. vaginalis-specifictarget-hybridizing sequence consists of the nucleotide sequence of SEQID NO:12; the first Eggerthella-specific target-hybridizing sequenceconsists of the nucleotide sequence of residues 28-51 of SEQ ID NO:17;and/or the second Eggerthella-specific target-hybridizing sequenceconsists of the nucleotide sequence of SEQ ID NO:16.

In some variations of a multiplex method as above, the firstLactobacillus-specific target-hybridizing sequence substantiallycorresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO:7,and the sample is further contacted with a third Lactobacillus-specificamplification oligomer for amplifying the Lactobacillus sp. targetregion, where the third Lactobacillus-specific amplification oligomercomprises a third Lactobacillus-specific target-hybridizing sequencesubstantially corresponding to the nucleotide sequence of residues 28-45of SEQ ID NO:8. In some such embodiments, the thirdLactobacillus-specific target-hybridizing sequence comprises thenucleotide sequence of residues 28-45 of SEQ ID NO:8. In a particularvariation, the third Lactobacillus-specific target-hybridizing sequenceconsists of the nucleotide sequence of residues 28-45 of SEQ ID NO:8.

In some embodiments of a multiplex method as above, at least one of thefirst Lactobacillus-specific amplification oligomer, the first G.vaginalis-specific amplification oligomer, and the firstEggerthella-specific amplification oligomer (at least one of the firstLactobacillus-specific amplification oligomer and the first G.vaginalis-specific amplification oligomer) is a promoter primer orpromoter provider further comprising a promoter sequence located 5′ tothe respective target hybridizing sequence. In some such embodimentswhere the first Lactobacillus-specific target-hybridizing sequencesubstantially corresponds to the nucleotide sequence of residues 28-45of SEQ ID NO:7 and the sample is further contacted with a thirdLactobacillus-specific amplification oligomer for amplifying theLactobacillus sp. target region, the third Lactobacillus-specificamplification oligomer is a promoter primer or promoter provider furthercomprising a promoter sequence located 5′ to the thirdLactobacillus-specific target hybridizing sequence. A particularlysuitable promoter sequence is a T7 promoter sequence such as, forexample, a promoter sequence having the nucleotide sequence of residues1-27 of SEQ ID NO:7. In specific variations, the firstLactobacillus-specific amplification oligomer has the nucleotidesequence of SEQ ID NO:7; the first G. vaginalis-specific amplificationoligomer has the nucleotide sequence of SEQ ID NO:13; and/or the firstEggerthella-specific amplification oligomer has the nucleotide sequenceof SEQ ID NO:17. In some embodiments as above where the sample isfurther contacted with a third Lactobacillus-specific amplificationoligomer for amplifying the Lactobacillus sp. target region, the thirdLactobacillus-specific amplification oligomer has the nucleotidesequence of SEQ ID NO:8.

In certain embodiments of a multiplex method as above, the methodfurther includes purifying the Lactobacillus, G. vaginalis, andEggerthella target nucleic acids, or purifying the Lactobacillus and G.vaginalis target nucleic acids, if present, from other components in thesample before step (2). In some such embodiments, the purifying stepincludes contacting the sample with at least one capture probe oligomercomprising a target-hybridizing sequence covalently attached to asequence or moiety that binds to an immobilized probe. For example, thesample may be contacted with a Lactobacillus-specific capture probeoligomer, a G. vaginalis-specific capture probe oligomer, and anEggerthella-specific capture probe oligomer, where each of theLactobacillus-specific, G. vaginalis-specific, and Eggerthella-specificcapture probe oligomers comprises a capture probe target-hybridizingsequence that specifically hybridizes to a target sequence within theLactobacillus, G. vaginalis, or Eggerthella target nucleic acid,respectively, and where each of the Lactobacillus-specific, G.vaginalis-specific, and Eggerthella-specific capture probetarget-hybridizing sequences is covalently attached to the sequence ormoiety that binds to the immobilized probe. In some embodiments directedto detection of Lactobacillus sp. and G. vaginalis, the sample may becontacted with a Lactobacillus-specific capture probe oligomer and a G.vaginalis-specific capture probe oligomer, where each of theLactobacillus-specific and G. vaginalis-specific capture probe oligomerscomprises a capture probe target-hybridizing sequence that specificallyhybridizes to a target sequence within the Lactobacillus or G. vaginalistarget nucleic acid, respectively, and where each of theLactobacillus-specific and G. vaginalis-specific capture probetarget-hybridizing sequences is covalently attached to the sequence ormoiety that binds to the immobilized probe. In specific variations, theLactobacillus-specific capture probe target-hybridizing sequencesubstantially corresponds to the nucleotide sequence of residues 1-12 ofSEQ ID NO:5, the G. vaginalis-specific capture probe target-hybridizingsequence substantially corresponds to the nucleotide sequence ofresidues 1-17 of SEQ ID NO:11, and/or the Eggerthella-specific captureprobe target hybridizing sequence substantially corresponds to thenucleotide sequence of residues 1-21 of SEQ ID NO:15. In someembodiments, the Lactobacillus-specific capture probe oligomer has thenucleotide sequence of SEQ ID NO:5, the G. vaginalis-specific captureprobe oligomer has the nucleotide sequence of SEQ ID NO:11, and/or theEggerthella-specific capture probe oligomer has the nucleotide sequenceof SEQ ID NO:15.

In some embodiments of a multiplex method as above, the detecting step(3) includes (i) contacting the one or more amplification products witha first Lactobacillus-specific detection probe that specificallyhybridizes to the Lactobacillus sp. target region, a first G.vaginalis-specific detection probe that specifically hybridizes to theG. vaginalis target region, and a first Eggerthella-specific detectionprobe that specifically hybridizes to the Eggerthella sp. target region,and (ii) detecting the presence or absence of any target-hybridizedLactobacillus-specific, G. vaginalis-specific, and/orEggerthella-specific detection probe. In some embodiments of a multiplexmethod for detection of Lactobacillus sp. and G. vaginalis as above, thedetecting step (3) includes (i) contacting the one or more amplificationproducts with a first Lactobacillus-specific detection probe thatspecifically hybridizes to the Lactobacillus sp. target region and afirst G. vaginalis-specific detection probe that specifically hybridizesto the G. vaginalis target region, and (ii) detecting the presence orabsence of any target-hybridized Lactobacillus-specific and/or G.vaginalis-specific detection probe. In some embodiments, the firstLactobacillus-specific detection probe comprises a target-hybridizingsequence substantially corresponding to SEQ ID NO:9, the first G.vaginalis-specific detection probe comprises a target-hybridizingsequence substantially corresponding to residues 1-19 of SEQ ID NO:14,and/or the first Eggerthella-specific detection probe comprises atarget-hybridizing sequence substantially corresponding to SEQ ID NO:18.In specific variations, the first Lactobacillus-specific detection probehas the nucleotide sequence of SEQ ID NO:9, the first G.vaginalis-specific detection probe has the nucleotide sequence of SEQ IDNO:14, and/or the first Eggerthella-specific detection probe has thenucleotide sequence of SEQ ID NO:18. In some embodiments as above, thedetecting step (3) further includes contacting the one or moreamplification products with a second Lactobacillus-specific detectionprobe that specifically hybridizes to the Lactobacillus sp. targetregion, where the second Lactobacillus-specific detection probecomprises a target-hybridizing sequence substantially corresponding toresidues 6-21 of SEQ ID NO:10; in some such embodiments, the secondLactobacillus-specific detection probe has the nucleotide sequence ofSEQ ID NO:10.

In certain embodiments of a multiplex method comprising the use of afirst Lactobacillus-specific detection probe, a first G.vaginalis-specific detection probe, and a first Eggerthella-specificdetection probe (or the use of a first Lactobacillus-specific detectionprobe and a first G. vaginalis-specific detection probe) as above, eachof the probes comprises a label. In some embodiments further includingcontacting the one or more amplification products with a secondLactobacillus-specific detection probe, the secondLactobacillus-specific detection probe comprises a label. Particularlysuitable labels include chemiluminescent and fluorescent labels.

In some embodiments of a multiplex method comprising the use of labeleddetection probes as above, the detecting step (3) occurs during theamplifying step (2). In some such variations, each detection probecomprises a fluorescent label and a quencher. Particularly suitabledetection probes comprising a fluorescent label and a quencher include amolecular torch, a molecular beacon, and a TaqMan detection probe.

In certain embodiments of a multiplex method comprising the use ofdetection probes as above, at least one of the firstLactobacillus-specific detection probe, the first G. vaginalis-specificdetection probe, and the first Eggerthella-specific detection probe (orat least one of the first Lactobacillus-specific detection probe and thefirst G. vaginalis-specific detection probe) further comprises anon-target-hybridizing sequence. In some embodiments further includingcontacting the one or more amplification products with a secondLactobacillus-specific detection probe, the secondLactobacillus-specific detection probe further comprises anon-target-hybridizing sequence. In some such variations as above, anyone of (e.g., each of) the detection probes is a molecular torch or amolecular beacon.

In some embodiments of a multiplex method as above, the amplificationreaction at step (2) is an isothermal amplification reaction. Inparticular variations, the isothermal amplification reaction is atranscription-mediated amplification (TMA) reaction. In certainembodiments, the isothermal amplification reaction is a real-timeamplification reaction.

In another aspect, the present invention provides an oligomercombination. In some embodiments, the oligomer combination is fordetermining the presence or absence of each of each of Lactobacillussp., G. vaginalis, and Eggerthella sp. in a sample. The oligomercombination generally includes the following oligomers:

-   -   (a) first and second Lactobacillus-specific amplification        oligomers for amplifying a target region of a Lactobacillus sp.        target nucleic acid, where (i) the first Lactobacillus-specific        amplification oligomer comprises a first Lactobacillus-specific        target-hybridizing sequence substantially corresponding to the        nucleotide sequence of residues 28-45 of SEQ ID NO:7 or residues        28-45 of SEQ ID NO:8 and (ii) the second Lactobacillus-specific        amplification oligomer comprises a second Lactobacillus-specific        target-hybridizing sequence substantially corresponding to the        nucleotide sequence of SEQ ID NO:6;    -   (b) first and second G. vaginalis-specific amplification        oligomers for amplifying a target region of a G. vaginalis        target nucleic acid, where (i) the first G. vaginalis-specific        amplification oligomer comprises a first G. vaginalis-specific        target-hybridizing sequence substantially corresponding to the        nucleotide sequence of residues 28-45 of SEQ ID NO:13 and (ii)        the second G. vaginalis-specific amplification oligomer        comprises a second G. vaginalis-specific target-hybridizing        sequence substantially corresponding to the nucleotide sequence        of SEQ ID NO:12; and    -   (c) first and second Eggerthella-specific amplification        oligomers for amplifying a target region of an Eggerthella sp.        target nucleic acid, where (i) the first Eggerthella-specific        amplification oligomer comprises a first Eggerthella-specific        target-hybridizing sequence substantially corresponding to the        nucleotide sequence of residues 28-51 of SEQ ID NO:17 and (ii)        the second Eggerthella-specific amplification oligomer comprises        a second Eggerthella-specific target-hybridizing sequence        substantially corresponding to the nucleotide sequence of SEQ ID        NO:16.

In other embodiments, the oligomer combination is for determining thepresence or absence of each of each of Lactobacillus sp. and G.vaginalis in a sample. The oligomer combination generally includes thefollowing oligomers:

-   -   (a) first and second Lactobacillus-specific amplification        oligomers for amplifying a target region of a Lactobacillus sp.        target nucleic acid, where (i) the first Lactobacillus-specific        amplification oligomer comprises a first Lactobacillus-specific        target-hybridizing sequence substantially corresponding to the        nucleotide sequence of residues 28-45 of SEQ ID NO:7 or residues        28-45 of SEQ ID NO:8 and (ii) the second Lactobacillus-specific        amplification oligomer comprises a second Lactobacillus-specific        target-hybridizing sequence substantially corresponding to the        nucleotide sequence of SEQ ID NO:6; and    -   (b) first and second G. vaginalis-specific amplification        oligomers for amplifying a target region of a G. vaginalis        target nucleic acid, where (i) the first G. vaginalis-specific        amplification oligomer comprises a first G. vaginalis-specific        target-hybridizing sequence substantially corresponding to the        nucleotide sequence of residues 28-45 of SEQ ID NO:13 and (ii)        the second G. vaginalis-specific amplification oligomer        comprises a second G. vaginalis-specific target-hybridizing        sequence substantially corresponding to the nucleotide sequence        of SEQ ID NO:12.

In some variations of an oligomer combination as above, the firstLactobacillus-specific target-hybridizing sequence comprises thenucleotide sequence of residues 28-45 of SEQ ID NO:7 or residues 28-45of SEQ ID NO:8; the second Lactobacillus-specific target-hybridizingsequence comprises the nucleotide sequence of SEQ ID NO:6; the first G.vaginalis-specific target-hybridizing sequence comprises the nucleotidesequence of residues 28-45 of SEQ ID NO:13; the second G.vaginalis-specific target-hybridizing sequence comprises the nucleotidesequence of SEQ ID NO:12; the first Eggerthella-specifictarget-hybridizing sequence comprises the nucleotide sequence ofresidues 28-51 of SEQ ID NO:17; and/or the second Eggerthella-specifictarget-hybridizing sequence comprises the nucleotide sequence of SEQ IDNO:16. In some such embodiments, the first Lactobacillus-specifictarget-hybridizing sequence consists of the nucleotide sequence ofresidues 28-45 of SEQ ID NO:7 or residues 28-45 of SEQ ID NO:8; thesecond Lactobacillus-specific target-hybridizing sequence consists ofthe nucleotide sequence of SEQ ID NO:6; the first G. vaginalis-specifictarget-hybridizing sequence consists of the nucleotide sequence ofresidues 28-45 of SEQ ID NO:13; the second G. vaginalis-specifictarget-hybridizing sequence consists of the nucleotide sequence of SEQID NO:12; the first Eggerthella-specific target-hybridizing sequenceconsists of the nucleotide sequence of residues 28-51 of SEQ ID NO:17;and/or the second Eggerthella-specific target-hybridizing sequenceconsists of the nucleotide sequence of SEQ ID NO:16.

In some variations of an oligomer combination as above, the firstLactobacillus-specific target-hybridizing sequence substantiallycorresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO:7,and the oligomer combination further includes a thirdLactobacillus-specific amplification oligomer for amplifying theLactobacillus sp. target region, where the third Lactobacillus-specificamplification oligomer comprises a third Lactobacillus-specifictarget-hybridizing sequence substantially corresponding to thenucleotide sequence of residues 28-45 of SEQ ID NO:8. In some suchembodiments, the third Lactobacillus-specific target-hybridizingsequence comprises the nucleotide sequence of residues 28-45 of SEQ IDNO:8. In a particular variation, the third Lactobacillus-specifictarget-hybridizing sequence consists of the nucleotide sequence ofresidues 28-45 of SEQ ID NO:8.

In some embodiments of an oligomer combination as above, at least one ofthe first Lactobacillus-specific amplification oligomer, the first G.vaginalis-specific amplification oligomer, and the firstEggerthella-specific amplification oligomer (or at least one of thefirst Lactobacillus-specific amplification oligomer and the first G.vaginalis-specific amplification oligomer) is a promoter primer orpromoter provider further comprising a promoter sequence located 5′ tothe respective target hybridizing sequence. In some such embodimentswhere the first Lactobacillus-specific target-hybridizing sequencesubstantially corresponds to the nucleotide sequence of residues 28-45of SEQ ID NO:7 and the sample is further contacted with a thirdLactobacillus-specific amplification oligomer for amplifying theLactobacillus sp. target region, the third Lactobacillus-specificamplification oligomer is a promoter primer or promoter provider furthercomprising a promoter sequence located 5′ to the thirdLactobacillus-specific target hybridizing sequence. A particularlysuitable promoter sequence is a T7 promoter sequence such as, forexample, a promoter sequence having the nucleotide sequence of residues1-27 of SEQ ID NO:7. In specific variations, the firstLactobacillus-specific amplification oligomer has the nucleotidesequence of SEQ ID NO:7; the first G. vaginalis-specific amplificationoligomer has the nucleotide sequence of SEQ ID NO:13; and/or the firstEggerthella-specific amplification oligomer has the nucleotide sequenceof SEQ ID NO:17. In some embodiments as above where the sample isfurther contacted with a third Lactobacillus-specific amplificationoligomer for amplifying the Lactobacillus sp. target region, the thirdLactobacillus-specific amplification oligomer has the nucleotidesequence of SEQ ID NO:8.

In certain embodiments of an oligomer combination as above, the oligomercombination further includes at least one capture probe oligomercomprising a target-hybridizing sequence covalently attached to asequence or moiety that binds to an immobilized probe. For example, theoligomer combination may include a Lactobacillus-specific capture probeoligomer, a G. vaginalis-specific capture probe oligomer, and anEggerthella-specific capture probe oligomer, where each of theLactobacillus-specific, G. vaginalis-specific, and Eggerthella-specificcapture probe oligomers comprises a capture probe target-hybridizingsequence that specifically hybridizes to a target sequence within theLactobacillus, G. vaginalis, or Eggerthella target nucleic acid,respectively, and where each of the Lactobacillus-specific, G.vaginalis-specific, and Eggerthella-specific capture probetarget-hybridizing sequences is covalently attached to the sequence ormoiety that binds to the immobilized probe. In some embodiments directedto an oligomer combination for determining the presence or absence ofeach of each of Lactobacillus sp. and G. vaginalis, the oligomercombination may include a Lactobacillus-specific capture probe oligomerand a G. vaginalis-specific capture probe oligomer, where each of theLactobacillus-specific and G. vaginalis-specific capture probe oligomerscomprises a capture probe target-hybridizing sequence that specificallyhybridizes to a target sequence within the Lactobacillus or G. vaginalistarget nucleic acid, respectively, and where each of theLactobacillus-specific and G. vaginalis-specific capture probetarget-hybridizing sequences is covalently attached to the sequence ormoiety that binds to the immobilized probe. In specific variations, theLactobacillus-specific capture probe target-hybridizing sequencesubstantially corresponds to the nucleotide sequence of residues 1-12 ofSEQ ID NO:5, the G. vaginalis-specific capture probe target-hybridizingsequence substantially corresponds to the nucleotide sequence ofresidues 1-17 of SEQ ID NO:11, and/or the Eggerthella-specific captureprobe target hybridizing sequence substantially corresponds to thenucleotide sequence of residues 1-21 of SEQ ID NO:15. In someembodiments, the Lactobacillus-specific capture probe oligomer has thenucleotide sequence of SEQ ID NO:5, the G. vaginalis-specific captureprobe oligomer has the nucleotide sequence of SEQ ID NO:11, and/or theEggerthella-specific capture probe oligomer has the nucleotide sequenceof SEQ ID NO:15.

In some embodiments of an oligomer combination as above, the oligomercombination further includes a first Lactobacillus-specific detectionprobe that specifically hybridizes to the Lactobacillus sp. targetregion, a first G. vaginalis-specific detection probe that specificallyhybridizes to the G. vaginalis target region, and a firstEggerthella-specific detection probe that specifically hybridizes to theEggerthella sp. target region. In some embodiments of an oligomercombination for determining the presence or absence of each of each ofLactobacillus sp. and G. vaginalis as above, the oligomer combinationfurther includes a first Lactobacillus-specific detection probe thatspecifically hybridizes to the Lactobacillus sp. target region and afirst G. vaginalis-specific detection probe that specifically hybridizesto the G. vaginalis target region. In some embodiments, the firstLactobacillus-specific detection probe comprises a target-hybridizingsequence substantially corresponding to SEQ ID NO:9, the first G.vaginalis-specific detection probe comprises a target-hybridizingsequence substantially corresponding to residues 1-19 of SEQ ID NO:14,and/or the first Eggerthella-specific detection probe comprises atarget-hybridizing sequence substantially corresponding to SEQ ID NO:18.In specific variations, the first Lactobacillus-specific detection probehas the nucleotide sequence of SEQ ID NO:9, the first G.vaginalis-specific detection probe has the nucleotide sequence of SEQ IDNO:14, and/or the first Eggerthella-specific detection probe has thenucleotide sequence of SEQ ID NO:18. In some embodiments as above, theoligomer combination further includes a second Lactobacillus-specificdetection probe that specifically hybridizes to the Lactobacillus sp.target region, where the second Lactobacillus-specific detection probecomprises a target-hybridizing sequence substantially corresponding toresidues 6-21 of SEQ ID NO:10; in some such embodiments, the secondLactobacillus-specific detection probe has the nucleotide sequence ofSEQ ID NO:10.

In certain embodiments of an oligomer combination comprising a firstLactobacillus-specific detection probe, a first G. vaginalis-specificdetection probe, and a first Eggerthella-specific detection probe (or afirst Lactobacillus-specific detection probe and a first G.vaginalis-specific detection probe) as above, each of the probescomprises a label. In some embodiments further including a secondLactobacillus-specific detection probe, the secondLactobacillus-specific detection probe comprises a label. Particularlysuitable labels include chemiluminescent and fluorescent labels.

In some embodiments of an oligomer combination comprising labeleddetection probes as above, each detection probe comprises a fluorescentlabel and a quencher. Particularly suitable detection probes comprisinga fluorescent label and a quencher include a molecular torch, amolecular beacon, and a TaqMan detection probe.

In certain embodiments of an oligomer combination comprising detectionprobes as above, at least one of the first Lactobacillus-specificdetection probe, the first G. vaginalis-specific detection probe, andthe first Eggerthella-specific detection probe (or at least one of thefirst Lactobacillus-specific detection probe and the first G.vaginalis-specific detection probe) further comprises anon-target-hybridizing sequence. In some embodiments further including asecond Lactobacillus-specific detection probe, the secondLactobacillus-specific detection probe further comprises anon-target-hybridizing sequence. In some such variations as above, anyone of (e.g., each of) the detection probes is a molecular torch or amolecular beacon.

In yet another aspect, the present invention provides a method fordetermining the presence or absence of Lactobacillus sp. in a sample.The method generally includes the following steps:

-   -   (1) contacting a sample, the sample suspected of containing        Lactobacillus sp., with first and second Lactobacillus-specific        amplification oligomers for amplifying a target region of a        Lactobacillus sp. target nucleic acid, where the first and        second Lactobacillus-specific amplification oligomers        respectively comprise first and second Lactobacillus-specific        target-hybridizing sequences, and where the first and second        amplification oligomers target a Lactobacillus sp. 16S rRNA        region corresponding to a region of SEQ ID NO:1 from about        nucleotide position 91 to about nucleotide position 265;    -   (2) performing an in vitro nucleic acid amplification reaction,        where any Lactobacillus sp. target nucleic acid, if present in        the sample, is used as a template for generating one or more        amplification products corresponding to the Lactobacillus sp.        target region; and    -   (3) detecting the presence or absence of the one or more        amplification products, thereby determining the presence or        absence of Lactobacillus sp. in the sample.

In some variations of a method for detecting Lactobacillus sp. as above,the first Lactobacillus-specific target-hybridizing sequencesubstantially corresponds to the nucleotide sequence of residues 28-45of SEQ ID NO:7 or residues 28-45 of SEQ ID NO:8, and/or the secondLactobacillus-specific target-hybridizing sequence substantiallycorresponds to the nucleotide sequence of SEQ ID NO:6. In some suchembodiments, the first Lactobacillus-specific target-hybridizingsequence comprises or consists of the nucleotide sequence of residues28-45 of SEQ ID NO:7 or residues 28-45 of SEQ ID NO:8, and/or the secondLactobacillus-specific target-hybridizing sequence comprises or consistsof the nucleotide sequence of SEQ ID NO:6.

In some embodiments of a method for detecting Lactobacillus sp. asabove, the first Lactobacillus-specific target-hybridizing sequencesubstantially corresponds to the nucleotide sequence of residues 28-45of SEQ ID NO:7, and the sample is further contacted with a thirdLactobacillus-specific amplification oligomer for amplifying theLactobacillus sp. target region, where the third Lactobacillus-specificamplification oligomer comprises a third Lactobacillus-specifictarget-hybridizing sequence substantially corresponding to thenucleotide sequence of residues 28-45 of SEQ ID NO:8. In some suchembodiments, the third Lactobacillus-specific target-hybridizingsequence comprises or consists of the nucleotide sequence of residues28-45 of SEQ ID NO:8.

In some embodiments of a method for detecting Lactobacillus sp. asabove, the first Lactobacillus-specific amplification oligomer furthercomprises a promoter sequence located 5′ to the firstLactobacillus-specific target hybridizing sequence. In some suchembodiments, where the first Lactobacillus-specific target-hybridizingsequence substantially corresponds to the nucleotide sequence ofresidues 28-45 of SEQ ID NO:7 and the sample is further contacted with athird Lactobacillus-specific amplification oligomer for amplifying theLactobacillus sp. target region, the third Lactobacillus-specificamplification oligomer is a promoter primer or promoter provider furthercomprising a promoter sequence located 5′ to the thirdLactobacillus-specific target hybridizing sequence. A particularlysuitable promoter sequence is a T7 promoter sequence such as, forexample, a promoter sequence having the nucleotide sequence of residues1-27 of SEQ ID NO:7. In specific variations, the firstLactobacillus-specific amplification oligomer has the nucleotidesequence of SEQ ID NO:7. In some embodiments as above where the sampleis further contacted with a third Lactobacillus-specific amplificationoligomer for amplifying the Lactobacillus sp. target region, the thirdLactobacillus-specific amplification oligomer has the nucleotidesequence of SEQ ID NO:8.

In certain embodiments of a method for detecting Lactobacillus sp. asabove, the method further includes purifying the Lactobacillus targetnucleic acid, if present, from other components in the sample beforestep (2). In some such embodiments, the purifying step includescontacting the sample with at least one capture probe oligomercomprising a target-hybridizing sequence covalently attached to asequence or moiety that binds to an immobilized probe. For example, thesample may be contacted with a Lactobacillus-specific capture probeoligomer comprising a capture probe target-hybridizing sequence thatspecifically hybridizes to a target sequence within the Lactobacillustarget nucleic acid, where the Lactobacillus-specific capture probetarget-hybridizing sequence is covalently attached to the sequence ormoiety that binds to the immobilized probe. In specific variations, theLactobacillus-specific capture probe target-hybridizing sequencesubstantially corresponds to the nucleotide sequence of residues 1-12 ofSEQ ID NO:5. In some embodiments, the Lactobacillus-specific captureprobe oligomer has the nucleotide sequence of SEQ ID NO:5.

In some embodiments of a method for detecting Lactobacillus sp. asabove, the detecting step (3) includes (i) contacting the one or moreamplification products with a first Lactobacillus-specific detectionprobe that specifically hybridizes to the Lactobacillus sp. targetregion, and (ii) detecting the presence or absence of anytarget-hybridized Lactobacillus-specific detection probe. In some suchembodiments, the first Lactobacillus-specific detection probe comprisesa target-hybridizing sequence substantially corresponding to SEQ IDNO:9. In a specific variation, the first Lactobacillus-specificdetection probe has the nucleotide sequence of SEQ ID NO:9. In someembodiments as above, the detecting step (3) further includes contactingthe one or more amplification products with a secondLactobacillus-specific detection probe that specifically hybridizes tothe Lactobacillus sp. target region, where the secondLactobacillus-specific detection probe comprises a target-hybridizingsequence substantially corresponding to residues 6-21 of SEQ ID NO:10;in some such embodiments, the second Lactobacillus-specific detectionprobe has the nucleotide sequence of SEQ ID NO:10.

In certain embodiments of a method for detecting Lactobacillus sp. asabove, where the method includes the use of a firstLactobacillus-specific detection probe, the first Lactobacillus-specificdetection probe comprises a label. In some embodiments further includingcontacting the one or more amplification products with a secondLactobacillus-specific detection probe, the secondLactobacillus-specific detection probe comprises a label. Particularlysuitable labels include chemiluminescent and fluorescent labels.

In some embodiments of a method for detecting Lactobacillus sp. asabove, where the method includes the use of a labeled detection probe,the detecting step (3) occurs during the amplifying step (2). In somesuch variations, the detection probe comprises a fluorescent label and aquencher. Particularly suitable detection probes comprising afluorescent label and a quencher include a molecular torch, a molecularbeacon, and a TaqMan detection probe.

In certain embodiments of a method for detecting Lactobacillus sp. asabove, where the method includes the use of a labeled detection probe,the first Lactobacillus-specific detection probe further comprises anon-target-hybridizing sequence. In some variations of the method, thefirst Lactobacillus-specific detection probe is a molecular torch or amolecular beacon.

In yet another aspect, the present invention provides a method fordetermining the presence or absence of G. vaginalis in a sample. Themethod generally includes the following steps:

-   -   (1) contacting a sample, the sample suspected of containing G.        vaginalis, with first and second G. vaginalis-specific        amplification oligomers for amplifying a target region of a G.        vaginalis target nucleic acid, where the first and second G.        vaginalis-specific amplification oligomers respectively comprise        first and second G. vaginalis-specific target hybridizing        sequences, and where the first and second amplification        oligomers target a G. vaginalis 16S rRNA region corresponding to        a region of SEQ ID NO:3 from about nucleotide position 964 to        about nucleotide position 1036;    -   (2) performing an in vitro nucleic acid amplification reaction,        where any G. vaginalis target nucleic acid, if present in the        sample, is used as a template for generating one or more        amplification products corresponding to the G. vaginalis target        region; and    -   (3) detecting the presence or absence of the one or more        amplification products, thereby determining the presence or        absence of G. vaginalis in the sample.

In some variations of a method for detecting G. vaginalis as above, thefirst G. vaginalis-specific target-hybridizing sequence substantiallycorresponds to the nucleotide sequence of residues 28-45 of SEQ IDNO:13, and/or the second G. vaginalis-specific target-hybridizingsequence substantially corresponds to the nucleotide sequence of SEQ IDNO:12. In some such embodiments, the first G. vaginalis-specifictarget-hybridizing sequence comprises or consists of the nucleotidesequence of residues 28-45 of SEQ ID NO:13, and/or the second G.vaginalis-specific target-hybridizing sequence comprises or consists ofthe nucleotide sequence of SEQ ID NO:12.

In some embodiments of a method for detecting G. vaginalis as above, thefirst G. vaginalis-specific amplification oligomer is a promoter primeror promoter provider further comprising a promoter sequence located 5′to the first G. vaginalis-specific target hybridizing sequence. Aparticularly suitable promoter sequence is a T7 promoter sequence suchas, for example, a promoter sequence having the nucleotide sequence ofresidues 1-27 of SEQ ID NO:7. In specific variations, the first G.vaginalis-specific amplification oligomer has the nucleotide sequence ofSEQ ID NO:13.

In certain embodiments of a method for detecting G. vaginalis as above,the method further includes purifying the G. vaginalis target nucleicacid, if present, from other components in the sample before step (2).In some embodiments, the purifying step further includes contacting thesample with at least one capture probe oligomer comprising atarget-hybridizing sequence covalently attached to a sequence or moietythat binds to an immobilized probe. For example, the sample may becontacted with a G. vaginalis-specific capture probe oligomer comprisinga capture probe target-hybridizing sequence that specifically hybridizesto a target sequence within the G. vaginalis target nucleic acid, wherethe G. vaginalis-specific capture probe target-hybridizing sequence iscovalently attached to the sequence or moiety that binds to theimmobilized probe. In specific variations, the G. vaginalis-specificcapture probe target-hybridizing sequence substantially corresponds tothe nucleotide sequence of residues 1-17 of SEQ ID NO:11. In someembodiments, the G. vaginalis-specific capture probe oligomer has thenucleotide sequence of SEQ ID NO:11.

In some embodiments of a method for detecting G. vaginalis as above, thedetecting step (3) includes (i) contacting the one or more amplificationproducts with a first G. vaginalis-specific detection probe thatspecifically hybridizes to the G. vaginalis target region, and (ii)detecting the presence or absence of any target-hybridized G.vaginalis-specific detection probe. In some such embodiments, the firstG. vaginalis-specific detection probe comprises a target-hybridizingsequence substantially corresponding to residues 1-19 of SEQ ID NO:14.In a specific variation, the first G. vaginalis-specific detection probehas the nucleotide sequence of SEQ ID NO:14.

In certain embodiments of a method for detecting G. vaginalis as above,the first G. vaginalis-specific detection probe detection probecomprises a label. Particularly suitable labels include chemiluminescentand fluorescent labels.

In some embodiments of a method for detecting G. vaginalis as above,where the method includes the use of a labeled detection probe, thedetecting step (3) occurs during the amplifying step (2). In some suchvariations, the detection probe comprises a fluorescent label and aquencher. Particularly suitable detection probes comprising afluorescent label and a quencher include a molecular torch, a molecularbeacon, or a TaqMan detection probe.

In certain embodiments of a method for detecting G. vaginalis as above,where the method includes the use of a labeled detection probe, thefirst G. vaginalis-specific detection probe further comprises anon-target-hybridizing sequence. In some variations of the method, thefirst G. vaginalis-specific detection probe is a molecular torch or amolecular beacon.

In still another aspect, the present invention provides a method fordetermining the presence or absence of Eggerthella sp. in a sample. Themethod generally includes the following steps:

-   -   (1) contacting a sample, the sample suspected of containing        Eggerthella sp., with first and second Eggerthella-specific        amplification oligomers for amplifying a target region of a        Eggerthella sp. target nucleic acid, where the first and second        Eggerthella-specific amplification oligomers respectively        comprise first and second Eggerthella-specific        target-hybridizing sequences, and where the first and second        amplification oligomers target a Eggerthella sp. 16S rRNA region        corresponding to a region of SEQ ID NO:4 from about nucleotide        position 165 to about nucleotide position 259;    -   (2) performing an in vitro nucleic acid amplification reaction,        wherein any Eggerthella sp. target nucleic acid, if present in        the sample, is used as a template for generating one or more        amplification products corresponding to the Eggerthella sp.        target region; and    -   (3) detecting the presence or absence of the one or more        amplification products, thereby determining the presence or        absence of Eggerthella sp. in the sample.

In some variations of a method for detecting Eggerthella sp. as above,the first Eggerthella-specific target-hybridizing sequence substantiallycorresponds to the nucleotide sequence of residues 28-51 of SEQ IDNO:17, and/or the second Eggerthella-specific target-hybridizingsequence substantially corresponds to the nucleotide sequence of SEQ IDNO:16. In some such embodiments, the first Eggerthella-specifictarget-hybridizing sequence comprises or consists of the nucleotidesequence of residues 28-51 of SEQ ID NO:17, and/or the secondEggerthella-specific target-hybridizing sequence comprises or consistsof the nucleotide sequence of SEQ ID NO:16.

In some embodiments of a method for detecting Eggerthella sp. as above,the first Eggerthella-specific amplification oligomer is a promoterprimer or promoter provider further comprising a promoter sequencelocated 5′ to the first Eggerthella-specific target hybridizingsequence. A particularly suitable promoter sequence is a T7 promotersequence such as, for example, a promoter sequence having the nucleotidesequence of residues 1-27 of SEQ ID NO:7. In specific variations, thefirst Eggerthella-specific amplification oligomer has the nucleotidesequence of SEQ ID NO:17.

In certain embodiments of a method for detecting Eggerthella sp. asabove, the method further includes purifying the Eggerthella sp. targetnucleic acid, if present, from other components in the sample beforestep (2). In some embodiments, the purifying step further includescontacting the sample with at least one capture probe oligomercomprising a target-hybridizing sequence covalently attached to asequence or moiety that binds to an immobilized probe. For example, thesample may be contacted with a Eggerthella-specific capture probeoligomer comprising a capture probe target-hybridizing sequence thatspecifically hybridizes to a target sequence within the Eggerthella sp.target nucleic acid, where the Eggerthella-specific capture probetarget-hybridizing sequence is covalently attached to the sequence ormoiety that binds to the immobilized probe. In specific variations, theEggerthella-specific capture probe target-hybridizing sequencesubstantially corresponds to the nucleotide sequence of residues 1-21 ofSEQ ID NO:15. In some embodiments, the Eggerthella-specific captureprobe oligomer has the nucleotide sequence of SEQ ID NO:15.

In some embodiments of a method for detecting Eggerthella sp. as above,the detecting step (3) includes (i) contacting the one or moreamplification products with a first Eggerthella-specific detection probethat specifically hybridizes to the Eggerthella sp. target region, and(ii) detecting the presence or absence of any target-hybridizedEggerthella-specific detection probe. In some such embodiments, thefirst Eggerthella-specific detection probe comprises atarget-hybridizing sequence substantially corresponding to SEQ ID NO:18.In a specific variation, the first Eggerthella-specific detection probehas the nucleotide sequence of SEQ ID NO:18.

In certain embodiments of a method for detecting Eggerthella sp. asabove, the first Eggerthella-specific detection probe detection probecomprises a label. Particularly suitable labels include chemiluminescentand fluorescent labels.

In some embodiments of a method for detecting Eggerthella sp. as above,where the method includes the use of a labeled detection probe, thedetecting step (3) occurs during the amplifying step (2). In some suchvariations, the detection probe comprises a fluorescent label and aquencher. Particularly suitable detection probes comprising afluorescent label and a quencher include a molecular torch, a molecularbeacon, or a TaqMan detection probe.

In certain embodiments of a method for detecting Eggerthella sp. asabove, where the method includes the use of a labeled detection probe,the first Eggerthella-specific detection probe further comprises anon-target-hybridizing sequence. In some variations of the method, thefirst Eggerthella-specific detection probe is a molecular torch or amolecular beacon.

In some embodiments of a method for detecting any one of Lactobacillussp., G. vaginalis, or Eggerthella sp. as above, the amplificationreaction at step (2) is an isothermal amplification reaction. Inparticular variations, the isothermal amplification reaction is atranscription-mediated amplification (TMA) reaction. In certainembodiments, the isothermal amplification reaction is a real-timeamplification reaction.

In still other aspects, the present invention provides an oligomercombination for determining the presence or absence of any one ofLactobacillus sp., G. vaginalis, and Eggerthella sp. in a sample. Invarious embodiments, the oligomer combination includes oligomers as setforth above for a method for determining the presence or absence ofLactobacillus sp., G. vaginalis, and Eggerthella sp. in a sample.

In yet other aspects, the present invention provides a composition or akit for determining the presence or absence of Bacterial Vaginosis (BV)in a subject. The composition or kit generally includes (a) a firstdetection probe that specifically hybridizes to a Lactobacillus sp.target nucleic acid, and (b) a second detection probe that specificallyhybridizes to a G. vaginalis target nucleic acid, where at least one ofthe first and second detection probes comprises a label, and where thecomposition or kit (i) does not comprise a detection probe thatspecifically hybridizes to a target nucleic acid from any fungal speciesand (ii) does not comprise a detection probe that specificallyhybridizes to a target nucleic acid from any bacterial species otherthan Lactobacillus sp. or G. vaginalis. In some embodiments, the firstdetection probe specifically hybridizes to at least one of an L. gasseritarget nucleic acid, an L. crispatus target nucleic acid, and an L.jensenii target nucleic acid. In some embodiments, the first detectionprobe targets a Lactobacillus sp. 16S rRNA region corresponding to aregion of SEQ ID NO:1 from about nucleotide position 91 to aboutnucleotide position 265, and/or the second detection probe targets a G.vaginalis 16S rRNA region corresponding to a region of SEQ ID NO:3 fromabout nucleotide position 964 to about nucleotide position 1036. Inparticular variations, the first detection probe comprises atarget-hybridizing sequence substantially corresponding to SEQ ID NO:9and/or the second detection probe comprises a target-hybridizingsequence substantially corresponding to residues 1-19 of SEQ ID NO:14;in some such embodiments, the first detection probe has the nucleotidesequence of SEQ ID NO:9 and/or the second detection probe has thenucleotide sequence of SEQ ID NO:14. In certain embodiments, thecomposition or kit further includes a third detection probe thatspecifically hybridizes to the Lactobacillus sp. target nucleic acid,e.g., a third detection probe comprising a target-hybridizing sequencethat substantially corresponds to residues 6-21 of SEQ ID NO:10. In aspecific variation, the third detection probe has the nucleotidesequence of SEQ ID NO:10.

These and other aspects of the invention will become evident uponreference to the following detailed description of the invention.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art pertinent to the methods and compositions described. As usedherein, the following terms and phrases have the meanings ascribed tothem unless specified otherwise.

The terms “a,” “an,” and “the” include plural referents, unless thecontext clearly indicates otherwise.

“Sample” includes any specimen that may contain Lactobacillus sp.,Gardnerella vaginalis, or Eggerthella sp. or components thereof, such asnucleic acids or fragments of nucleic acids. Samples include “biologicalsamples” which include any tissue or material derived from a living ordead human that may contain Lactobacillus sp., Gardnerella vaginalis, orEggerthella sp. or components thereof (e.g., a target nucleic acidderived therefrom), including, e.g., vaginal swab samples, cervicalbrush samples, respiratory tissue or exudates such as bronchoscopy,bronchoalveolar lavage (BAL) or lung biopsy, sputum, saliva, peripheralblood, plasma, serum, lymph node, gastrointestinal tissue, feces, urine,semen or other body fluids or materials. The biological sample may betreated to physically or mechanically disrupt tissue or cell structure,thus releasing intracellular components into a solution which mayfurther contain enzymes, buffers, salts, detergents and the like, whichare used to prepare, using standard methods, a biological sample foranalysis. Also, samples may include processed samples, such as thoseobtained from passing samples over or through a filtering device, orfollowing centrifugation, or by adherence to a medium, matrix, orsupport.

“Nucleic acid” refers to a multimeric compound comprising two or morecovalently bonded nucleosides or nucleoside analogs having nitrogenousheterocyclic bases, or base analogs, where the nucleosides are linkedtogether by phosphodiester bonds or other linkages to form apolynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNApolymers or oligonucleotides, and analogs thereof. A nucleic acid“backbone” may be made up of a variety of linkages, including one ormore of sugar-phosphodiester linkages, peptide-nucleic acid bonds (in“peptide nucleic acids” or PNAs, see PCT No. WO 95/32305),phosphorothioate linkages, methylphosphonate linkages, or combinationsthereof. Sugar moieties of the nucleic acid may be either ribose ordeoxyribose, or similar compounds having known substitutions, e.g., 2′methoxy substitutions and 2′ halide substitutions (e.g., 2′-F).Nitrogenous bases may be conventional bases (A, G, C, T, U), analogsthereof (e.g., inosine, 5-methylisocytosine, isoguanine; TheBiochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11^(th) ed.,1992, Abraham et al., 2007, BioTechniques 43: 617-24), which includederivatives of purine or pyrimidine bases (e.g., N⁴-methyldeoxygaunosine, deaza- or aza-purines, deaza- or aza-pyrimidines,pyrimidine bases having substituent groups at the 5 or 6 position,purine bases having an altered or replacement substituent at the 2, 6and/or 8 position, such as 2-amino-6-methylaminopurine,O⁶-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines,4-dimethylhydrazine-pyrimidines, and O⁴-alkyl-pyrimidines, andpyrazolo-compounds, such as unsubstituted or 3-substitutedpyrazolo[3,4-d]pyrimidine; U.S. Pat. Nos. 5,378,825, 6,949,367 and PCTNo. WO 93/13121). Nucleic acids may include “abasic” residues in whichthe backbone does not include a nitrogenous base for one or moreresidues (U.S. Pat. No. 5,585,481). A nucleic acid may comprise onlyconventional sugars, bases, and linkages as found in RNA and DNA, or mayinclude conventional components and substitutions (e.g., conventionalbases linked by a 2′ methoxy backbone, or a nucleic acid including amixture of conventional bases and one or more base analogs). Nucleicacids may include “locked nucleic acids” (LNA), in which one or morenucleotide monomers have a bicyclic furanose unit locked in an RNAmimicking sugar conformation, which enhances hybridization affinitytoward complementary sequences in single-stranded RNA (ssRNA),single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) (Vester etal., Biochemistry 43:13233-41, 2004). Nucleic acids may include modifiedbases to alter the function or behavior of the nucleic acid, e.g.,addition of a 3′-terminal dideoxynucleotide to block additionalnucleotides from being added to the nucleic acid. Synthetic methods formaking nucleic acids in vitro are well-known in the art although nucleicacids may be purified from natural sources using routine techniques.

The term “polynucleotide,” as used herein, denotes a nucleic acid chain.Throughout this application, nucleic acids are designated by the5′-terminus to the 3′-terminus. Standard nucleic acids, e.g., DNA andRNA, are typically synthesized “5′-to-3′,” i.e., by the addition ofnucleotides to the 3′-terminus of a growing nucleic acid.

A “nucleotide,” as used herein, is a subunit of a nucleic acidconsisting of a phosphate group, a 5-carbon sugar and a nitrogenousbase. The 5-carbon sugar found in RNA is ribose. In DNA, the 5-carbonsugar is 2′-deoxyribose. The term also includes analogs of suchsubunits, such as a methoxy group at the 2′ position of the ribose(2′-O-Me).

A “nucleic-acid-based detection assay,” as used herein, is an assay forthe detection of a target sequence within a target nucleic acid andutilizing one more oligonucleotides that specifically hybridize to thetarget sequence.

In certain embodiments in accordance with the present invention, anucleic-acid-based detection assay is an “amplification-based assay,”i.e., an assay that utilizes one or more steps for amplifying a nucleicacid target sequence. Various amplification methods for use in detectionassays are known in the art, several of which are summarized furtherherein. For the sake of clarity, an amplification-based assay mayinclude one or more steps that do not amplify a target sequence, suchas, for example, steps used in non-amplification-based assay methods(e.g., a hybridization assay or a cleavage-based assay).

In other embodiments, a nucleic-acid-based detection assay is a“non-amplification-based assay,” i.e., an assay that does not rely onany step for amplifying a nucleic acid target sequence. For the sake ofclarity, a nucleic-acid-based detection assay that includes a reactionfor extension of a primer in the absence of any corresponding downstreamamplification oligomer (e.g., extension of a primer by a reversetranscriptase to generate an RNA:DNA duplex followed by an RNasedigestion of the RNA, resulting in a single-stranded cDNA complementaryto an RNA target but without generating copies of the cDNA) isunderstood to be a non-amplification-based assay.

An exemplary non-amplification-based assay is a “cleavage-based assay,”which is an assay that relies on the specific cleavage, by a flapendonuclease, of a linear duplex cleavage structure formed by thespecific hybridization of overlapping oligonucleotides to a targetnucleic acid. In these assays, a probe oligonucleotide containing anon-target-hybridizing flap region is cleaved in an overlap-dependentmanner by the flap endonuclease to release a cleavage product that isthen detected. The principles of cleavage-based assays are well-known inthe art, and exemplary assays are described in, for example, Lyamichevet al. (Nat. Biotechnol. 17:292-296, 1999), Ryan et al. (Mol. Diagn.4:135-144, 1999), Allawi et al. (J. Chn. Microbiol. 44:3443-3447, 2006),U.S. Pat. Nos. 5,846,717 & 6,706,471 to Brow et al., and U.S. Pat. No.5,614,402 to Dahlberg et al. Cleavage-based assays include, e.g., thecommercially available Invader® assays (Hologic, Inc., Madison, Wis.).

A “target nucleic acid,” as used herein, is a nucleic acid comprising atarget sequence to be detected. Target nucleic acids may be DNA or RNAas described herein, and may be either single-stranded ordouble-stranded. The target nucleic acid may include other sequencesbesides the target sequence.

By “isolated” it is meant that a sample containing a target nucleic acidis taken from its natural milieu, but the term does not connote anydegree of purification.

The term “target sequence,” as used herein, refers to the particularnucleotide sequence of a target nucleic acid that is to be detected. The“target sequence” includes the complexing sequences to whicholigonucleotides (e.g., probe oligonucleotide, priming oligonucleotidesand/or promoter oligonucleotides) complex during a detection process(e.g., an amplification-based detection assay such as, for example, TMAor PCR, or a non-amplification-based detection assay such as, forexample, a cleavage-based assay). Where the target nucleic acid isoriginally single-stranded, the term “target sequence” will also referto the sequence complementary to the “target sequence” as present in thetarget nucleic acid. Where the target nucleic acid is originallydouble-stranded, the term “target sequence” refers to both the sense (+)and antisense (−) strands. In choosing a target sequence, the skilledartisan will understand that a “unique” sequence should be chosen so asto distinguish between unrelated or closely related target nucleicacids.

“Target-hybridizing sequence” is used herein to refer to the portion ofan oligomer that is configured to hybridize with a target nucleic acidsequence. Preferably, the target-hybridizing sequences are configured tospecifically hybridize with a target nucleic acid sequence.Target-hybridizing sequences may be 100% complementary to the portion ofthe target sequence to which they are configured to hybridize, but notnecessarily. Target-hybridizing sequences may also include inserted,deleted and/or substituted nucleotide residues relative to a targetsequence. Less than 100% complementarity of a target-hybridizingsequence to a target sequence may arise, for example, when the targetnucleic acid is a plurality strains within a species, such as would bethe case for an oligomer configured to hybridize to the various strainsof Lactobacillus. It is understood that other reasons exist forconfiguring a target-hybridizing sequence to have less than 100%complementarity to a target nucleic acid.

The term “targets a sequence,” as used herein in reference to a regionof Lactobacillus sp., G. vaginalis, or Eggerthella sp. nucleic acid,refers to a process whereby an oligonucleotide hybridizes to the targetsequence in a manner that allows for detection as described herein. Inone embodiment, the oligonucleotide is complementary with the targetedLactobacillus sp., G. vaginalis, or Eggerthella sp. nucleic acidsequence and contains no mismatches. In another embodiment, theoligonucleotide is complementary but contains 1, 2, 3, 4, or 5mismatches with the targeted Lactobacillus sp., G. vaginalis, orEggerthella sp. nucleic acid sequence. Preferably, the oligonucleotidethat hybridizes to the target nucleic acid sequence includes at least 10to as many as 50 nucleotides complementary to the target sequence. It isunderstood that at least 10 and as many as 50 is an inclusive range suchthat 10, 50 and each whole number there between are included.Preferably, the oligomer specifically hybridizes to the target sequence.

The term “configured to” denotes an actual arrangement of thepolynucleotide sequence configuration of a referenced oligonucleotidetarget-hybridizing sequence. For example, oligonucleotides that areconfigured to specifically hybridize to a target sequence have apolynucleotide sequence that specifically hybridizes to the referencedsequence under stringent hybridization conditions.

The term “configured to specifically hybridize to” as used herein meansthat the target-hybridizing region of an oligonucleotide is designed tohave a polynucleotide sequence that could target a sequence of thereferenced Lactobacillus sp., G. vaginalis, or Eggerthella sp. targetregion. Such an oligonucleotide is not limited to targeting thatsequence only, but is rather useful as a composition, in a kit or in amethod for targeting a Lactobacillus sp., G. vaginalis, or Eggerthellasp. target nucleic acid. The oligonucleotide is designed to function asa component of an assay for detection of Lactobacillus sp., G.vaginalis, or Eggerthella sp. from a sample, and therefore is designedto target Lactobacillus sp., G. vaginalis, or Eggerthella sp. in thepresence of other nucleic acids commonly found in testing samples.“Specifically hybridize to” does not mean exclusively hybridize to, assome small level of hybridization to non-target nucleic acids may occur,as is understood in the art. Rather, “specifically hybridize to” meansthat the oligonucleotide is configured to function in an assay toprimarily hybridize the target so that an accurate detection of targetnucleic acid in a sample can be determined. The term “configured to”denotes an actual arrangement of the polynucleotide sequenceconfiguration of the oligonucleotide target-hybridizing sequence.

The term “fragment,” as used herein in reference to an Lactobacillussp., G. vaginalis, or Eggerthella sp. targeted nucleic acid, refers to apiece of contiguous nucleic acid. In certain embodiments, the fragmentincludes contiguous nucleotides from an Lactobacillus sp., G. vaginalis,or Eggerthella sp. 16S ribosomal RNA, wherein the number of 16Scontiguous nucleotides in the fragment are less than that for the entire16S.

The term “region,” as used herein, refers to a portion of a nucleic acidwherein said portion is smaller than the entire nucleic acid. Forexample, when the nucleic acid in reference is an oligonucleotidepromoter primer, the term “region” may be used refer to the smallerpromoter portion of the entire oligonucleotide. Similarly, and also asexample only, when the nucleic acid is a 16S ribosomal RNA, the term“region” may be used to refer to a smaller area of the nucleic acid,wherein the smaller area is targeted by one or more oligonucleotides ofthe invention. As another non-limiting example, when the nucleic acid inreference is an amplicon, the term region may be used to refer to thesmaller nucleotide sequence identified for hybridization by thetarget-hybridizing sequence of a probe.

The interchangeable terms “oligomer,” “oligo,” and “oligonucleotide”refer to a nucleic acid having generally less than 1,000 nucleotide (nt)residues, including polymers in a range having a lower limit of about 5nt residues and an upper limit of about 500 to 900 nt residues. In someembodiments, oligonucleotides are in a size range having a lower limitof about 12 to 15 nt and an upper limit of about 50 to 600 nt, and otherembodiments are in a range having a lower limit of about 15 to 20 nt andan upper limit of about 22 to 100 nt. Oligonucleotides may be purifiedfrom naturally occurring sources or may be synthesized using any of avariety of well-known enzymatic or chemical methods. The termoligonucleotide does not denote any particular function to the reagent;rather, it is used generically to cover all such reagents describedherein. An oligonucleotide may serve various different functions. Forexample, it may function as a primer if it is specific for and capableof hybridizing to a complementary strand and can further be extended inthe presence of a nucleic acid polymerase; it may function as a primerand provide a promoter if it contains a sequence recognized by an RNApolymerase and allows for transcription (e.g., a T7 Primer); and it mayfunction to detect a target nucleic acid if it is capable of hybridizingto the target nucleic acid, or an amplicon thereof, and further providesa detectible moiety (e.g., an acridinium-ester compound).

As used herein, an oligonucleotide “substantially corresponding to” aspecified reference nucleic acid sequence means that the oligonucleotideis sufficiently similar to the reference nucleic acid sequence such thatthe oligonucleotide has similar hybridization properties to thereference nucleic acid sequence in that it would hybridize with the sametarget nucleic acid sequence under stringent hybridization conditions.One skilled in the art will understand that “substantially correspondingoligonucleotides” can vary from a reference sequence and still hybridizeto the same target nucleic acid sequence. It is also understood that afirst nucleic acid corresponding to a second nucleic acid includes theRNA and DNA thereof and includes the complements thereof, unless thecontext clearly dictates otherwise. This variation from the nucleic acidmay be stated in terms of a percentage of identical bases within thesequence or the percentage of perfectly complementary bases between theprobe or primer and its target sequence. Thus, in certain embodiments,an oligonucleotide “substantially corresponds” to a reference nucleicacid sequence if these percentages of base identity or complementarityare from 100% to about 80%. In preferred embodiments, the percentage isfrom 100% to about 85%. In more preferred embodiments, this percentageis from 100% to about 90%; in other preferred embodiments, thispercentage is from 100% to about 95%. Similarly, a region of a nucleicacid or amplified nucleic acid can be referred to herein ascorresponding to a reference nucleic acid sequence. One skilled in theart will understand the various modifications to the hybridizationconditions that might be required at various percentages ofcomplementarity to allow hybridization to a specific target sequencewithout causing an unacceptable level of non-specific hybridization.

An “amplification oligomer” is an oligomer, at least the 3′-end of whichis complementary to a target nucleic acid, and which hybridizes to atarget nucleic acid, or its complement, and participates in a nucleicacid amplification reaction. An example of an amplification oligomer isa “primer” that hybridizes to a target nucleic acid and contains a 3′ OHend that is extended by a polymerase in an amplification process.Another example of an amplification oligomer is an oligomer that is notextended by a polymerase (e.g., because it has a 3′ blocked end) butparticipates in or facilitates amplification. For example, the 5′ regionof an amplification oligonucleotide may include a promoter sequence thatis non-complementary to the target nucleic acid (which may be referredto as a “promoter primer” or “promoter provider”). Those skilled in theart will understand that an amplification oligomer that functions as aprimer may be modified to include a 5′ promoter sequence, and thusfunction as a promoter primer. Incorporating a 3′ blocked end furthermodifies the promoter primer, which is now capable of hybridizing to atarget nucleic acid and providing an upstream promoter sequence thatserves to initiate transcription, but does not provide a primer foroligo extension. Such a modified oligo is referred to herein as a“promoter provider” oligomer. Size ranges for amplificationoligonucleotides include those that are about 10 to about 70 nt long(not including any promoter sequence or poly-A tails) and contain atleast about 10 contiguous bases, or even at least 12 contiguous basesthat are complementary to a region of the target nucleic acid sequence(or a complementary strand thereof). The contiguous bases are at least80%, or at least 90%, or completely complementary to the target sequenceto which the amplification oligomer binds. An amplification oligomer mayoptionally include modified nucleotides or analogs, or additionalnucleotides that participate in an amplification reaction but are notcomplementary to or contained in the target nucleic acid, or templatesequence. It is understood that when referring to ranges for the lengthof an oligonucleotide, amplicon, or other nucleic acid, that the rangeis inclusive of all whole numbers (e.g., 19-25 contiguous nucleotides inlength includes 19, 20, 21, 22, 23, 24 & 25).

As used herein, a “promoter” is a specific nucleic acid sequence that isrecognized by a DNA-dependent RNA polymerase (“transcriptase”) as asignal to bind to the nucleic acid and begin the transcription of RNA ata specific site.

As used herein, a “promoter provider” or “provider” refers to anoligonucleotide comprising first and second regions, and which ismodified to prevent the initiation of DNA synthesis from its3′-terminus. The “first region” of a promoter provider oligonucleotidecomprises a base sequence which hybridizes to a DNA template, where thehybridizing sequence is situated 3′, but not necessarily adjacent to, apromoter region. The hybridizing portion of a promoter oligonucleotideis typically at least 10 nucleotides in length, and may extend up to 50or more nucleotides in length. The “second region” comprises a promotersequence for an RNA polymerase. A promoter oligonucleotide is engineeredso that it is incapable of being extended by an RNA- or DNA-dependentDNA polymerase, e.g., reverse transcriptase, preferably comprising ablocking moiety at its 3′-terminus as described above. As referred toherein, a “T7 Provider” is a blocked promoter provider oligonucleotidethat provides an oligonucleotide sequence that is recognized by T7 RNApolymerase.

“Amplification” refers to any known procedure for obtaining multiplecopies of a target nucleic acid sequence or its complement or fragmentsthereof. The multiple copies may be referred to as amplicons oramplification products. Known amplification methods include both thermalcycling and isothermal amplification methods. In some embodiments,isothermal amplification methods are preferred. Replicase-mediatedamplification, polymerase chain reaction (PCR), ligase chain reaction(LCR), strand-displacement amplification (SDA), andtranscription-mediated or transcription-associated amplification arenon-limiting examples of nucleic acid amplification methods.Replicase-mediated amplification uses self-replicating RNA molecules,and a replicase such as QB-replicase (e.g., U.S. Pat. No. 4,786,600).PCR amplification uses a DNA polymerase, pairs of primers, and thermalcycling to synthesize multiple copies of two complementary strands ofdsDNA or from a cDNA (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and4,800,159). LCR amplification uses four or more differentoligonucleotides to amplify a target and its complementary strand byusing multiple cycles of hybridization, ligation, and denaturation(e.g., U.S. Pat. No. 5,427,930 and U.S. Pat. No. 5,516,663). SDA uses aprimer that contains a recognition site for a restriction endonucleaseand an endonuclease that nicks one strand of a hemimodified DNA duplexthat includes the target sequence, whereby amplification occurs in aseries of primer extension and strand displacement steps (e.g., U.S.Pat. No. 5,422,252; U.S. Pat. No. 5,547,861; and U.S. Pat. No.5,648,211). Preferred embodiments use an amplification method suitablefor the amplification of RNA target nucleic acids, such astranscription-mediated amplification (TMA) or NASBA, but it will beapparent to persons of ordinary skill in the art that oligomersdisclosed herein may be readily used as primers in other amplificationmethods.

“Transcription-associated amplification,” also referred to herein as“transcription-mediated amplification” (TMA), refers to nucleic acidamplification that uses an RNA polymerase to produce multiple RNAtranscripts from a nucleic acid template. These methods generally employan RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates,ribonucleoside triphosphates, and a template complementaryoligonucleotide that includes a promoter sequence, and optionally mayinclude one or more other oligonucleotides. TMA methods are embodimentsof amplification methods used for amplifying and detecting HSV targetsequences as described herein. Variations of transcription-associatedamplification are well-known in the art as previously disclosed indetail (e.g., U.S. Pat. Nos. 4,868,105; 5,124,246; 5,130,238; 5,399,491;5,437,990; 5,554,516; and 7,374,885; and PCT Pub. Nos. WO 88/01302, WO88/10315, and WO 95/03430). The person of ordinary skill in the art willappreciate that the disclosed compositions may be used in amplificationmethods based on extension of oligomer sequences by a polymerase.

As used herein, the term “real-time TMA” refers to single-primertranscription-mediated amplification (“TMA”) of target nucleic acid thatis monitored by real-time detection means.

The term “amplicon,” which is used interchangeably with “amplificationproduct,” refers to the nucleic acid molecule generated during anamplification procedure that is complementary or homologous to asequence contained within the target sequence. These terms can be usedto refer to a single strand amplification product, a double strandamplification product or one of the strands of a double strandamplification product.

“Probe,” “detection probe,” “detection oligonucleotide,” and “detectionprobe oligomer” are used interchangeably herein to refer to a nucleicacid oligomer that hybridizes specifically to a target sequence in anucleic acid, or in an amplified nucleic acid, under conditions thatpromote hybridization to allow detection of the target sequence oramplified nucleic acid. Detection may either be direct (e.g., a probehybridized directly to its target sequence) or indirect (e.g., a probelinked to its target via an intermediate molecular structure). Probesmay be DNA, RNA, analogs thereof or combinations thereof and they may belabeled or unlabeled. A probe's “target sequence” generally refers to asmaller nucleic acid sequence within a larger nucleic acid sequence thathybridizes specifically to at least a portion of a probe oligomer bystandard base pairing. A probe may comprise target-specific sequencesand other sequences that contribute to the three-dimensionalconformation of the probe (e.g., U.S. Pat. Nos. 5,118,801; 5,312,728;6,849,412; 6,835,542; 6,534,274; and 6,361,945; and US Pub. No.20060068417). In a preferred embodiment, the detection probe comprises a2′ methoxy backbone which can result in a higher signal being obtained.

The term “TaqMan® probe” refers to detection oligonucleotides thatcontain a fluorescent dye, typically on the 5′ base, and anon-fluorescent quenching dye (quencher), typically on the 3′ base. Whenirradiated, the excited fluorescent dye transfers energy to the nearbyquenching dye molecule rather than fluorescing, resulting in anon-fluorescent substrate. During amplification, the exonucleaseactivity of the polymerase cleaves the TaqMan probe to separate thefluorophore from the quencher, thereby allowing an unquenched signal tobe emitted from the fluorophore as an indicator of amplification.

As used herein, a “label” refers to a moiety or compound joined directlyor indirectly to a probe that is detected or leads to a detectablesignal. Direct labelling can occur through bonds or interactions thatlink the label to the probe, including covalent bonds or non-covalentinteractions, e.g., hydrogen bonds, hydrophobic and ionic interactions,or formation of chelates or coordination complexes. Indirect labellingcan occur through use of a bridging moiety or “linker” such as a bindingpair member, an antibody or additional oligomer, which is eitherdirectly or indirectly labeled, and which may amplify the detectablesignal. Labels include any detectable moiety, such as a radionuclide,ligand (e.g., biotin, avidin), enzyme or enzyme substrate, reactivegroup, or chromophore (e.g., dye, particle, or bead that impartsdetectable color), luminescent compound (e.g., bioluminescent,phosphorescent, or chemiluminescent labels), or fluorophore. Labels maybe detectable in a homogeneous assay in which bound labeled probe in amixture exhibits a detectable change different from that of an unboundlabeled probe, e.g., instability or differential degradation properties.A “homogeneous detectable label” can be detected without physicallyremoving bound from unbound forms of the label or labeled probe (e.g.,U.S. Pat. Nos. 5,283,174, 5,656,207, and 5,658,737). Labels includechemiluminescent compounds, e.g., acridinium ester (“AE”) compounds thatinclude standard AE and derivatives (e.g., U.S. Pat. Nos. 5,656,207,5,658,737, and 5,639,604). Synthesis and methods of attaching labels tonucleic acids and detecting labels are well known (e.g., Sambrook etal., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989), Chapter 10; U.S. Pat.Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333). Morethan one label, and more than one type of label, may be present on aparticular probe, or detection may use a mixture of probes in which eachprobe is labeled with a compound that produces a detectable signal(e.g., U.S. Pat. Nos. 6,180,340 and 6,350,579).

As used herein, structures referred to as “molecular torches” aredesigned to include distinct regions of self-complementarity (“theclosing domain”) which are connected by a joining region (“the targetbinding domain”) and which hybridize to one another under predeterminedhybridization assay conditions. All or part of the nucleotide sequencescomprising target closing domains may also function as target bindingdomains. Thus, target closing sequences can include, target bindingsequences, non-target binding sequences, and combinations thereof.

“Capture probe,” “capture oligonucleotide,” “target captureoligonucleotide,” and “capture probe oligomer” are used interchangeablyherein to refer to a nucleic acid oligomer that specifically hybridizesto a target sequence in a target nucleic acid by standard base pairingand joins to a binding partner on an immobilized probe to capture thetarget nucleic acid to a support. One example of a capture oligomerincludes an oligonucleotide comprising two binding regions: a targethybridizing sequence and an immobilized probe-binding region. Avariation of this example, the two regions may be present on twodifferent oligomers joined together by one or more linkers. Anotherembodiment of a capture oligomer the target hybridizing sequence is asequence that includes random or non-random poly-GU, poly-GT, or poly Usequences to bind non-specifically to a target nucleic acid and link itto an immobilized probe on a support (see, e.g., PCT Pub No. WO2008/016988). The immobilized probe binding region can be a nucleic acidsequence, referred to as a tail. Tails include a substantiallyhomopolymeric tail of about 10 to 40 nucleotides (e.g., A₁₀ to A₄₀), orof about 14 to 33 nt (e.g., T₃A₁₄ to T₃A₃₀), that bind to acomplementary immobilized sequence attached to the support particle orsupport matrix. Thus, a non-limiting example of preferred nucleic acidtails can in some embodiments include T₀₋₄A₁₀₋₄₀ sequences. Anotherexample of a capture oligomer comprises two regions, a targethybridizing sequence and a binding pair member that is not a nucleicacid sequence.

As used herein, an “immobilized oligonucleotide,” “immobilized probe” or“immobilized nucleic acid” refers to a nucleic acid binding partner thatjoins a capture oligomer to a support, directly or indirectly. Animmobilized probe joined to a support facilitates separation of acapture probe bound target from unbound material in a sample. Oneembodiment of an immobilized probe is an oligomer joined to a supportthat facilitates separation of bound target sequence from unboundmaterial in a sample. Supports may include known materials, such asmatrices and particles free in solution, which may be made ofnitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene,silane, polypropylene, metal, or other compositions, of which oneembodiment is magnetically attractable particles. Supports may bemonodisperse magnetic spheres (e.g., uniform size±5%), to which animmobilized probe is joined directly (via covalent linkage, chelation,or ionic interaction), or indirectly (via one or more linkers), wherethe linkage or interaction between the probe and support is stableduring hybridization conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A illustrates a reference sequence (SEQ ID NO:1) for Lactobacillus16S ribosomal RNA gene (Lactobacillus crispatus strain ATCC 33820 16Sribosomal RNA gene, partial sequence, found at GenBank under accessionnumber NR_041800.1 GI:343201103). FIG. 1B illustrates a referencesequence (SEQ ID NO:88) for Lactobacillus jensenii 16S ribosomal RNAgene, partial sequence, GenBank accession number NR_025087.1GI:219857499.

FIG. 2 illustrates a reference sequence (SEQ ID NO:2) for Lactobacillus16S ribosomal RNA gene (Lactobacillus gasseri partial 16S rRNA gene,type strain CIP 102991Tstrain, found at GenBank under accession numberHE573914.1 GI:341599788).

FIG. 3 illustrates a reference sequence (SEQ ID NO:3) for Gardnerallavaginalis 16S ribosomal RNA gene (Gardneralla vaginalis strain 594 16Sribosomal RNA gene, complete sequence, found at GenBank under accessionnumber NR_044694.2 GI:545589071).

FIG. 4 illustrates a reference sequence (SEQ ID NO:4) for Eggerthella16S ribosomal RNA gene (Uncultured Eggerthella sp. clone 123-f2 68 16Sribosomal RNA gene, partial sequence, found at GenBank under accessionnumber AY738656.1 GI:52222145).

DESCRIPTION

The present invention is generally directed to methods and compositionsfor determining the presence or absence of select bacterial organisms ina sample. In some embodiments, the present invention provides methodsand compositions for diagnosing Bacterial Vaginosis (BV) in a subject.In other, non-mutually exclusive embodiments, the present inventionprovides methods for the detection of any one or more of Lactobacillussp., G. vaginalis, and Eggerthella sp. in a sample, where the methodincludes performing amplification-based detection of a 16S rRNA targetnucleic from one or more of Lactobacillus sp., G. vaginalis, andEggerthella sp. The present invention further provides compositions(including reaction mixtures) and kits comprising a combination ofoligomers for detecting any one or more of Lactobacillus sp., G.vaginalis, and Eggerthella sp. in a sample. The oligomer combinationgenerally includes at least two amplification oligomers for detectingone or more of Lactobacillus sp., G. vaginalis, and Eggerthella sp. in asample, and may further include one or more additional oligomers asdescribed herein for performing amplification-based detection ofLactobacillus sp., G. vaginalis, and/or Eggerthella sp. such as, e.g., acapture probe and/or a detection probe.

The methods for diagnosing BV generally include detecting the presenceor absence of one or more of Lactobacillus sp., G. vaginalis, andEggerthella sp. in a sample from a subject suspected of having BV. Inparticular, an assay is performed for the specific detection in thesample of each of Lactobacillus sp., G. vaginalis, and Eggerthella sp.,or the specific detection in the sample of each of Lactobacillus sp. andG. vaginalis. Based on the results from the detection assay, a status ofeither positive or negative is assigned for each of Lactobacillus sp.,G. vaginalis, and Eggerthella sp., or each of Lactobacillus sp. and G.vaginalis, and the presence or absence of BV in the subject isdetermined based on a combination of the assigned Lactobacillus sp.status, G. vaginalis status, and Eggerthella sp. status, or based on acombination of the assigned Lactobacillus sp. status and G. vaginalisstatus.

In particular, where a status of either positive or negative is assignedfor each of Lactobacillus sp., G. vaginalis, and Eggerthella sp. anegative status for both G. vaginalis and Eggerthella sp. indicates theabsence of BV in the subject, and a positive status for both G.vaginalis and Eggerthella sp. indicates the presence of BV in thesubject. Further, if the status of Lactobacillus sp. is positive, then anegative status for at least one of G. vaginalis and Eggerthellaindicates the absence of BV in the subject, and if the status ofLactobacillus sp. is negative, then a positive status for at least oneof G. vaginalis and Eggerthella indicates the presence of BV in thesubject. Table 1 below shows a BV indication matrix based on thecombined status for Lactobacillus, G. vaginalis, and Eggerthella.

TABLE 1 Bacterial Vaginosis Indication Matrix Based on Lactobacillus, G.vaginalis, and Eggerthella Lactobacillus status G. vaginalis statusEggerthella status BV + + + Yes + + − No + − + No − − − No − + − Yes −− + Yes − + + Yes + − − No

Alternatively, where a status of either positive or negative is assignedfor each of Lactobacillus sp. and G. vaginalis, a negative status for G.vaginalis indicates the absence of BV in the subject. Further, if thestatus of G. vaginalis is positive, then a positive status forLactobacillus sp. indicates the absence of BV in the subject and anegative status for Lactobacillus sp. indicates the presence of BV inthe subject. It is noted, though, that the presence of a high copynumber (e.g., ≥1.4e10 copies/mL) of G. vaginalis in a sample is apositive indicator of BV in the subject. Table 2 below shows a BVindication matrix based on the combined status for Lactobacillus and G.vaginalis.

TABLE 2 Bacterial Vaginosis Indication Matrix Based on Lactobacillus andG. vaginalis Lactobacillus status G. vaginalis status BV + + No + − No− + Yes − − No

While Lactobacillus sp., G. vaginalis, and/or Eggerthella sp. may bedetected using any suitable method, it is presently preferred that thesebacteria are detected using a nucleic-acid-based detection assay.Nucleic-acid-based detection assays in accordance with the presentinvention generally utilize oligonucleotides that specifically hybridizeto a target nucleic acid of Lactobacillus sp., G. vaginalis, orEggerthella sp. with minimal cross-reactivity to other nucleic acidssuspected of being in a sample. Accordingly, oligonucleotides fornucleic-acid-based detection of the select species of Lactobacillus sp.,G. vaginalis, or Eggerthella sp. will have minimal cross-reactivity tospecies within other bacterial genera, including, for example,Trichomonas sp.; Trichomonas vaginalis; Candida sp.; Bacterium from theorder Clostridiales; Clostridium-like sp.; Atopobium sp.; Atopobiumvaginae; Enterobacteria; Peptostreptococcus micros; Aerococcuschristensenii; Leptotrichia amnionii; Peptoniphilus sp.; Dialister sp.;Mycoplasma hominis; Sneathia sanguinegens; Anaerococcus tetradius;Mobiluncus sp.; Mobiluncus hominis; Megasphaera sp.; Prevotella sp.;Leptotrichia sanguinegens; and Finegoldia magna. In one aspect, anucleic-acid-based detection assay in accordance with the presentinvention further includes components for detecting one of more of theseorganisms, or other bacterial genera associated with BV.

In particular embodiments, a nucleic-acid-based detection assay targetsthe 16S rRNA of Lactobacillus sp., G. vaginalis, and/or Eggerthella sp.,or a gene encoding the 16S rRNA. Particularly suitable target regions ofthe 16S rRNA or the encoding gene are (i) a Lactobacillus sp. 16S rRNAregion corresponding to a region of SEQ ID NO:1 from about nucleotideposition 91 to about nucleotide position 265; (ii) a Lactobacillus sp.16S rRNA region corresponding to a region of SEQ ID NO:2 from aboutnucleotide position 90 to about nucleotide position 263; (iii) a G.vaginalis 16S rRNA region corresponding to a region of SEQ ID NO:3 fromabout nucleotide position 964 to about nucleotide position 1036; and(iv) an Eggerthella sp. 16S rRNA region corresponding to a region of SEQID NO:4 from about nucleotide position 165 to about nucleotide position259. In specific variations of a nucleic-acid-based detection assaytargeting a 16S rRNA region as above, (a) a Lactobacillus-specificoligonucleotide includes a target-hybridizing region comprising asequence substantially corresponding to the sequence shown in SEQ IDNO:6, a sequence substantially corresponding to the sequence shown inresidues 28-45 of SEQ ID NO:7, a sequence substantially corresponding tothe sequence shown in residues 28-45 of SEQ ID NO:8, a sequencesubstantially corresponding to the sequence shown in SEQ ID NO:9, or asequence substantially corresponding to the sequence shown in residues6-21 of SEQ ID NO:10; (b) a G. vaginalis-specific oligonucleotideincludes a target-hybridizing region comprising a sequence substantiallycorresponding to the sequence shown in SEQ ID NO:12, a sequencesubstantially corresponding to the sequence shown in residues 28-45 ofSEQ ID NO:13, or a sequence substantially corresponding to the sequenceshown in residues 1-19 of SEQ ID NO:14; and/or (c) anEggerthella-specific oligonucleotide includes a target-hybridizingregion comprising a sequence substantially corresponding to the sequenceshown in SEQ ID NO:16, a sequence substantially corresponding to thesequence shown in residues 28-51 of SEQ ID NO:17, or a sequencesubstantially corresponding to the sequence shown SEQ ID NO:18. In somesuch embodiments, (a) a Lactobacillus-specific oligonucleotide includesa target-hybridizing region comprising or consisting of the sequenceshown in SEQ ID NO:6, the sequence shown in residues 28-45 of SEQ IDNO:7, the sequence shown in residues 28-45 of SEQ ID NO:8, or thesequence shown in SEQ ID NO:9; (b) a G. vaginalis-specificoligonucleotide includes a target-hybridizing region comprising orconsisting of the sequence shown in SEQ ID NO:12, the sequence shown inresidues 28-45 of SEQ ID NO:13, or the sequence shown in residues 1-19of SEQ ID NO:14; and/or (c) an Eggerthella-specific oligonucleotideincludes a target-hybridizing region comprising or consisting of thesequence shown in SEQ ID NO:16, the sequence shown in residues 28-51 ofSEQ ID NO:17, or the sequence shown SEQ ID NO:18. In certainembodiments, a nucleic-acid-based detection assay utilizes at least twoor three Lactobacillus-specific oligonucleotides, at least two or threeG. vaginalis-specific oligonucleotides, and/or at least two or threeEggerthella-specific oligonucleotides, which may be oligonucleotidesselected from those specified above.

In some embodiments of a method comprising the use of anucleic-acid-base detection assay, an amplification-based assay is usedto detect Lactobacillus sp., G. vaginalis, and/or Eggerthella sp. Suchvariations generally include amplifying a target sequence within abacterial target nucleic acid utilizing an in vitro nucleic acidamplification reaction and detecting the amplified product by, forexample, specifically hybridizing the amplified product with a nucleicacid detection probe that provides a signal to indicate the presence ofa bacterial target in the sample. The amplification step includescontacting the sample with two or more amplification oligomers specificfor a target sequence in a target nucleic acid (e.g., a target sequencein a 16S rRNA) to produce an amplified product if the target nucleicacid is present in the sample. Amplification synthesizes additionalcopies of the target sequence or its complement such as, e.g., by usingat least one nucleic acid polymerase to extend the sequence from anamplification oligomer (a primer) using a template strand. Oneembodiment for detecting the amplified product uses a hybridizing stepthat includes contacting the amplified product with at least one probespecific for a sequence amplified by the selected amplificationoligomers, e.g., a sequence contained in the target sequence flanked bya pair of selected amplification oligomers. Suitable amplificationmethods include, for example, replicase-mediated amplification,polymerase chain reaction (PCR), ligase chain reaction (LCR),strand-displacement amplification (SDA), and transcription-mediated ortranscription-associated amplification (TMA). Such amplification methodsare well-known in the art (see, e.g., discussion of amplificationmethods in Definitions section, supra) and are readily used inaccordance with the methods of the present invention.

For example, some amplification methods that use TMA amplificationinclude the following steps. Briefly, the target nucleic acid thatcontains the sequence to be amplified is provided as single strandednucleic acid (e.g., ssRNA or ssDNA). Those skilled in the art willappreciate that conventional melting of double stranded nucleic acid(e.g., dsDNA) may be used to provide single-stranded target nucleicacids. A promoter primer binds specifically to the target nucleic acidat its target sequence and a reverse transcriptase (RT) extends the 3′end of the promoter primer using the target strand as a template tocreate a cDNA copy of the target sequence strand, resulting in anRNA:DNA duplex. An RNase digests the RNA strand of the RNA:DNA duplexand a second primer binds specifically to its target sequence, which islocated on the cDNA strand downstream from the promoter primer end. RTsynthesizes a new DNA strand by extending the 3′ end of the secondprimer using the first cDNA template to create a dsDNA that contains afunctional promoter sequence. An RNA polymerase specific for thepromoter sequence then initiates transcription to produce RNAtranscripts that are about 100 to 1000 amplified copies (“amplicons”) ofthe initial target strand in the reaction. Amplification continues whenthe second primer binds specifically to its target sequence in each ofthe amplicons and RT creates a DNA copy from the amplicon RNA templateto produce an RNA:DNA duplex. RNase in the reaction mixture digests theamplicon RNA from the RNA:DNA duplex and the promoter primer bindsspecifically to its complementary sequence in the newly synthesized DNA.RT extends the 3′ end of the promoter primer to create a dsDNA thatcontains a functional promoter to which the RNA polymerase binds totranscribe additional amplicons that are complementary to the targetstrand. The autocatalytic cycles of making more amplicon copies repeatduring the course of the reaction resulting in about a billion-foldamplification of the target nucleic acid present in the sample. Theamplified products may be detected in real-time during amplification, orat the end of the amplification reaction by using a probe that bindsspecifically to a target sequence contained in the amplified products.Detection of a signal resulting from the bound probes indicates thepresence of the target nucleic acid in the sample.

In some embodiments, the method utilizes a “reverse” TMA reaction. Insuch variations, the initial or “forward” amplification oligomer is apriming oligonucleotide that hybridizes to the target nucleic acid inthe vicinity of the 3′-end of the target region. A reverse transcriptase(RT) synthesizes a cDNA strand by extending the 3′-end of the primerusing the target nucleic acid as a template. The second or “reverse”amplification oligomer is a promoter primer or promoter provider havinga target-hybridizing sequence configure to hybridize to atarget-sequence contained within the synthesized cDNA strand. Where thesecond amplification oligomer is a promoter primer, RT extends the 3′end of the promoter primer using the cDNA strand as a template to createa second, cDNA copy of the target sequence strand, thereby creating adsDNA that contains a functional promoter sequence. Amplification thencontinues essentially as described above for initiation of transcriptionfrom the promoter sequence utilizing an RNA polymerase. Alternatively,where the second amplification oligomer is a promoter provider, aterminating oligonucleotide, which hybridizes to a target sequence thatis in the vicinity to the 5′-end of the target region, is typicallyutilized to terminate extension of the priming oligomer at the 3′-end ofthe terminating oligonucleotide, thereby providing a defined 3′-end forthe initial cDNA strand synthesized by extension from the primingoligomer. The target-hybridizing sequence of the promoter provider thenhybridizes to the defined 3′-end of the initial cDNA strand, and the3′-end of the cDNA strand is extended to add sequence complementary tothe promoter sequence of the promoter provider, resulting in theformation of a double-stranded promoter sequence. The initial cDNAstrand is then used a template to transcribe multiple RNA transcriptscomplementary to the initial cDNA strand, not including the promoterportion, using an RNA polymerase that recognizes the double-strandedpromoter and initiates transcription therefrom. Each of these RNAtranscripts is then available to serve as a template for furtheramplification from the first priming amplification oligomer.

In certain embodiments comprising an amplification-based detectionassay, a combination of at least two amplification oligomers is utilizedfor the detection of a Lactobacillus sp. 16S rRNA or a gene encoding aLactobacillus sp. 16S rRNA. The oligomer combination may include firstand second amplification oligomers for amplifying a Lactobacillus sp.nucleic acid target region corresponding to a region of SEQ ID NO:1 fromabout nucleotide position 91 to about nucleotide position 265, and/or aLactobacillus sp. nucleic acid target region corresponding to a regionof SEQ ID NO:2 from about nucleotide position 90 to about nucleotideposition 263. For example, in some embodiments, the first amplificationoligomer includes a target-hybridizing sequence substantiallycorresponding to the nucleotide sequence of residues 28-45 of SEQ IDNO:7 or residues 28-45 of SEQ ID NO:8, and/or the second amplificationoligomer includes a target-hybridizing sequence substantiallycorresponding to the nucleotide sequence of SEQ ID NO:6. In moreparticular variations, the first amplification oligomer includes atarget-hybridizing sequence comprising or consisting of the nucleotidesequence of residues 28-45 of SEQ ID NO:7 or residues 28-45 of SEQ IDNO:8, and/or the second amplification oligomer includes atarget-hybridizing sequence comprising or consisting of the nucleotidesequence of SEQ ID NO:6. In some embodiments, where the firstamplification oligomer includes a target-hybridizing sequencesubstantially corresponding to the nucleotide sequence of residues 28-45of SEQ ID NO:7, the amplification detection assay further utilizes athird amplification oligomer that includes a target-hybridizing sequencesubstantially corresponding to the nucleotide sequence of residues 28-45of SEQ ID NO:8; in some such variations, the third amplificationoligomer includes a target-hybridizing sequence comprising or consistingof the nucleotide sequence of residues 28-45 of SEQ ID NO:8. In someembodiments as above, at least one amplification is a promoter primer orpromoter provide further comprising a promoter sequence located 5′ tothe respective target-hybridizing sequence (e.g., a T7 promotersequences such as, for example, the nucleotide sequence of residues 1-27of SEQ ID NO:7); in some such embodiments, the first amplificationoligomer is a promoter primer or promoter provider, and/or, if present,the third amplification oligomer is a promoter primer or promoterprovider. In more specific variations, the first amplification oligomerconsists of the nucleotide sequence of SEQ ID NO:7, the secondamplification oligomer consists of the nucleotide sequence of SEQ IDNO:6, and/or, if present, the third amplification oligomer consists ofthe nucleotide sequence of SEQ ID NO:8.

In certain embodiments comprising an amplification-based detectionassay, a combination of at least two amplification oligomers is utilizedfor the detection of a G. vaginalis 16S rRNA or a gene encoding a G.vaginalis 16S rRNA. The oligomer combination may include first andsecond amplification oligomers for amplifying a G. vaginalis nucleicacid target region corresponding to a region of SEQ ID NO:3 from aboutnucleotide position 964 to about nucleotide position 1036. For example,in some embodiments, the first amplification oligomer includes atarget-hybridizing sequence substantially corresponding to thenucleotide sequence of residues 28-45 of SEQ ID NO:13 and/or the secondamplification oligomer includes a target-hybridizing sequencesubstantially corresponding to the nucleotide sequence of SEQ ID NO:12.In more particular variations, the first amplification oligomer includesa target-hybridizing sequence comprising or consisting of the nucleotidesequence of residues 28-45 of SEQ ID NO:13 and/or the secondamplification oligomer includes a target-hybridizing sequence comprisingor consisting of the nucleotide sequence of SEQ ID NO:12. hi someembodiments as above, at least one amplification is a promoter primer orpromoter provide further comprising a promoter sequence located 5′ tothe respective target-hybridizing sequence (e.g., a T7 promotersequences such as, for example, the nucleotide sequence of residues 1-27of SEQ ID NO:7); in some such embodiments, the first amplificationoligomer is a promoter primer or promoter provider. In more specificvariations, the first amplification oligomer consists of the nucleotidesequence of SEQ ID NO:13 and/or the second amplification oligomerconsists of the nucleotide sequence of SEQ ID NO:12.

In certain embodiments comprising an amplification-based detectionassay, a combination of at least two amplification oligomers is utilizedfor the detection of an Eggerthella sp. 16S rRNA or a gene encoding anEggerthella sp. 16S rRNA. The oligomer combination may include first andsecond amplification oligomers for amplifying an Eggerthella sp. nucleicacid target region corresponding to a region of SEQ ID NO:4 from aboutnucleotide position 165 to about nucleotide position 259. For example,in some embodiments, the first amplification oligomer includes atarget-hybridizing sequence substantially corresponding to thenucleotide sequence of residues 28-51 of SEQ ID NO:17 and/or the secondamplification oligomer includes a target-hybridizing sequencesubstantially corresponding to the nucleotide sequence of SEQ ID NO:16.In more particular variations, the first amplification oligomer includesa target-hybridizing sequence comprising or consisting of the nucleotidesequence of residues 28-51 of SEQ ID NO:17 and/or the secondamplification oligomer includes a target-hybridizing sequence comprisingor consisting of the nucleotide sequence of SEQ ID NO:16. In someembodiments as above, at least one amplification is a promoter primer orpromoter provide further comprising a promoter sequence located 5′ tothe respective target-hybridizing sequence (e.g., a T7 promotersequences such as, for example, the nucleotide sequence of residues 1-27of SEQ ID NO:7); in some such embodiments, the first amplificationoligomer is a promoter primer or promoter provider. In more specificvariations, the first amplification oligomer consists of the nucleotidesequence of SEQ ID NO:17 and/or the second amplification oligomerconsists of the nucleotide sequence of SEQ ID NO:16.

Detection of the amplified products may be accomplished by a variety ofmethods to detect a signal specifically associated with the amplifiedtarget sequence. The nucleic acids may be associated with a surface thatresults in a physical change, such as a detectable electrical change.Amplified nucleic acids may be detected by concentrating them in or on amatrix and detecting the nucleic acids or dyes associated with them(e.g., an intercalating agent such as ethidium bromide or cyber green),or detecting an increase in dye associated with nucleic acid in solutionphase. Other methods of detection may use nucleic acid detection probesthat are configured to specifically hybridize to a sequence in theamplified product and detecting the presence of the probe:productcomplex, or by using a complex of probes that may amplify the detectablesignal associated with the amplified products (e.g., U.S. Pat. Nos.5,424,413; 5,451,503; and 5,849,481; each incorporated by referenceherein). Directly or indirectly labeled probes that specificallyassociate with the amplified product provide a detectable signal thatindicates the presence of the target nucleic acid in the sample. Forexample, if the target nucleic acid is the 16S rRNA of Lactobacillussp., G. vaginalis, and/or Eggerthella sp., the amplified product willcontain a target sequence in or complementary to a sequence in the 16SrRNA, and a probe will bind directly or indirectly to a sequencecontained in the amplified product to indicate the presence of the 16SrRNA of Lactobacillus sp., G. vaginalis, and/or Eggerthella sp. in thetested sample.

Detection probes that hybridize to the complementary amplified sequencesmay be DNA or RNA oligomers, or oligomers that contain a combination ofDNA and RNA nucleotides, or oligomers synthesized with a modifiedbackbone, e.g., an oligomer that includes one or more 2′-methoxysubstituted ribonucleotides. Probes used for detection of the amplifiedsequences may be unlabeled and detected indirectly (e.g., by binding ofanother binding partner to a moiety on the probe) or may be labeled witha variety of detectable labels. In some embodiments of the method fordiagnosing BV, such as in certain embodiments usingtranscription-mediated amplification (TMA), the detection probe is alinear chemiluminescently labeled probe such as, e.g., a linearacridinium ester (AE) labeled probe.

The detection step may also provide additional information on theamplified sequence, such as, e.g., all or a portion of its nucleic acidbase sequence. Detection may be performed after the amplificationreaction is completed, or may be performed simultaneously withamplifying the target region, e.g., in real time. In one embodiment, thedetection step allows homogeneous detection, e.g., detection of thehybridized probe without removal of unhybridized probe from the mixture(see, e.g., U.S. Pat. Nos. 5,639,604 and 5,283,174, each incorporated byreference herein).

In embodiments that detect the amplified product near or at the end ofthe amplification step, a linear detection probe may be used to providea signal to indicate hybridization of the probe to the amplifiedproduct. One example of such detection uses a luminescentally labeledprobe that hybridizes to target nucleic acid. Luminescent label is thenhydrolyzed from non-hybridized probe. Detection is performed bychemiluminescence using a luminometer. (see, e.g., International PatentApplication Pub. No. WO 89/002476, incorporated by reference herein). Inother embodiments that use real-time detection, the detection probe maybe a hairpin probe such as, for example, a molecular beacon, moleculartorch, or hybridization switch probe that is labeled with a reportermoiety that is detected when the probe binds to amplified product. Suchprobes may comprise target-hybridizing sequences andnon-target-hybridizing sequences. Various forms of such probes have beendescribed previously (see, e.g., U.S. Pat. Nos. 5,118,801; 5,312,728;5,925,517; 6,150,097; 6,849,412; 6,835,542; 6,534,274; and 6,361,945;and US Patent Application Pub. Nos. 20060068417A1 and 20060194240A1;each incorporated by reference herein).

In certain embodiments comprising an amplification-based detection assaytargeting a Lactobacillus sp., G. vaginalis, and/or Eggerthella sp. 16SrRNA or a gene encoding a Lactobacillus sp., G. vaginalis, and/orEggerthella sp. 16S rRNA, the method utilizes one or more detectionprobes that specifically hybridizes to a Lactobacillus sp., G.vaginalis, and/or Eggerthella sp. 16S rRNA amplification product. Inparticular variations, a Lactobacillus-specific detection probespecifically hybridizes to a nucleic acid target region corresponding toa region of SEQ ID NO:1 from about nucleotide position 91 to aboutnucleotide position 265, and/or a Lactobacillus sp. nucleic acid targetregion corresponding to a region of SEQ ID NO:2 from about nucleotideposition 90 to about nucleotide position 263; a G. vaginalis-specificdetection probe specifically hybridizes to a nucleic acid target regioncorresponding to a region of SEQ ID NO:3 from about nucleotide position964 to about nucleotide position 1036; and/or an Eggerthella-specificprobe specifically hybridizes to a nucleic acid target regioncorresponding to a region of SEQ ID NO:4 from about nucleotide position165 to about nucleotide position 259. For example, in some variations, aprobe for detection of a Lactobacillus sp. amplification productincludes a target-hybridizing sequence substantially corresponding tothe sequence of SEQ ID NO:9 or a sequence substantially corresponding tothe sequence of residues 6-21 of SEQ ID NO:10 (e.g., a probe thatcomprises the target-hybridizing sequence of SEQ ID NO:9 or residues6-21 of SEQ ID NO:10). In some variations, a probe for detection of a G.vaginalis amplification product includes a target-hybridizing sequencesubstantially corresponding to the sequence of residues 1-19 of SEQ IDNO:14 (e.g., a probe that comprises the target-hybridizing sequence ofresidues 1-19 of SEQ ID NO:14). In some variations, a probe fordetection of an Eggerthella sp. amplification product includes atarget-hybridizing sequence substantially corresponding to the sequenceof SEQ ID NO:18 (e.g., a probe that comprises the target-hybridizingsequence of SEQ ID NO:18). In certain embodiments, a probe for detectionof a Lactobacillus sp. amplification product comprises or consists ofthe sequence of SEQ ID NO:9 or SEQ ID NO:10; a probe for detection of aG. vaginalis amplification product comprises or consists of the sequenceof SEQ ID NO:14; and/or a probe for detection of an Eggerthella sp.amplification product comprises or consists of the sequence of SEQ IDNO:18.

In some embodiments of a method comprising the use of anucleic-acid-base detection assay, a non-amplification-based assay isused to detect Lactobacillus sp., G. vaginalis, and/or Eggerthella sp.In some such embodiments, the non-amplification-based assay is ahybridization assay comprising the hybridization of a specific detectionprobe to a target nucleic acid. Methods for conducting polynucleotidehybridization assays have been well developed in the art. Hybridizationassay procedures and conditions will vary depending on the applicationand are selected in accordance with the general binding methods known,including those referred to in, e.g., Maniatis et al., MolecularCloning: A Laboratory Manual (3rd ed. Cold Spring Harbor, N.Y., 2002),and Berger and Kimmel, Methods in Enzymology, Vol. 152, Guide toMolecular Cloning Techniques (Academic Press, Inc., San Diego, Calif.,1987). Generally, the probe and sample are mixed under conditions thatwill permit specific nucleic acid hybridization, and specifichybridization of the probe to its respective target is then detected.Nucleic acid hybridization is adaptable to a variety of assay formats.One suitable format is the sandwich assay format, which is particularlyadaptable to hybridization under non-denaturing conditions. A primarycomponent of a sandwich-type assay is a solid support, which hasadsorbed to it or covalently coupled to it immobilized nucleic acidprobe that is unlabeled and complementary to one portion of the DNAsequence. Target nucleic acid is hybridized to the immobilized probe,and a second, labeled detection probe—which is complementary to a secondand different region of the same DNA strand to which the immobilized,unlabeled nucleic acid probe is hybridized—is hybridized to the [targetnucleic acid]:[immobilized probe] duplex to detect the target nucleicacid. Another exemplary format utilizes electrochemical detection oftarget nucleic acids hybridized to unlabeled detection probesimmobilized on a suitable electrode surface as a signal transducer. See,e.g., Drummond et al., Nat. Biotechnol. 21:1192, 2003; Gooding,Electroanalysis 14:1149, 2002; Wang, Anal. Chim. Acta 469:63, 2002;Cagnin et al., Sensors 9:3122, 2009; Katz and Willner, Electroanalysis15:913, 2003; Daniels and Pourmand, Electroanalysis 19:1239, 2007.

In certain embodiments comprising a hybridization assay, a detectionprobe is utilized for the detection of a Lactobacillus sp., G.vaginalis, and/or Eggerthella sp. 16S rRNA or a gene encoding aLactobacillus sp., G. vaginalis, and/or Eggerthella sp. 16S rRNA. Insuch embodiments, a probe for detecting a Lactobacillus sp. 16S rRNA orgene encoding a Lactobacillus sp. 16S rRNA specifically hybridizes to anucleic acid target region corresponding to a region of SEQ ID NO:1 fromabout nucleotide position 91 to about nucleotide position 265, and/or aLactobacillus sp. nucleic acid target region corresponding to a regionof SEQ ID NO:2 from about nucleotide position 90 to about nucleotideposition 263; a probe for detecting a G. vaginalis 16S rRNA or geneencoding a G. vaginalis 16S rRNA specifically hybridizes to a nucleicacid target region corresponding to a region of SEQ ID NO:3 from aboutnucleotide position 964 to about nucleotide position 1036; and/or aprobe for detecting an Eggerthella sp. 16S rRNA or gene encoding anEggerthella sp. 16S rRNA specifically hybridizes to a nucleic acidtarget region corresponding to a region of SEQ ID NO:4 from aboutnucleotide position 165 to about nucleotide position 259. For example,in some variations, a probe for detection of a Lactobacillus sp. 16SrRNA or gene encoding a Lactobacillus sp. 16S rRNA includes atarget-hybridizing sequence substantially corresponding to the sequenceof SEQ ID NO:9 or a sequence substantially corresponding to the sequenceof residues 6-21 of SEQ ID NO:10 (e.g., a probe that comprises thetarget-hybridizing sequence of SEQ ID NO:9 or residues 6-21 of SEQ IDNO:10). In some variations, a probe for detection of a G. vaginalis 16SrRNA or gene encoding a G. vaginalis 16S rRNA includes atarget-hybridizing sequence substantially corresponding to the sequenceof residues 1-19 of SEQ ID NO:14 (e.g., a probe that comprises thetarget-hybridizing sequence of residues 1-19 of SEQ ID NO:14). In somevariations, a probe for detection of an Eggerthella sp. 16S rRNA or geneencoding an Eggerthella sp. 16S rRNA includes a target-hybridizingsequence substantially corresponding to the sequence of SEQ ID NO:18(e.g., a probe that comprises the target-hybridizing sequence of SEQ IDNO:18). In certain embodiments, a probe for detection of a Lactobacillussp. 16S rRNA or gene encoding a Lactobacillus sp. 16S rRNA comprises orconsists of the sequence of SEQ ID NO:9 or SEQ ID NO:10; a probe fordetection of a G. vaginalis 16S rRNA or gene encoding a G. vaginalis sp.16S rRNA comprises or consists of the sequence of SEQ ID NO:14; and/or aprobe for detection of an Eggerthella sp. 16S rRNA or gene encoding anEggerthella sp. 16S rRNA comprises or consists of the sequence of SEQ IDNO:18.

In some embodiments, a non-amplification-based assay for detection ofLactobacillus sp., G. vaginalis, and/or Eggerthella sp. is acleavage-based assay, in which a probe oligonucleotide containing anon-target-hybridizing flap region is cleaved in an overlap-dependentmanner by a flap endonuclease to release a cleavage product that is thendetected. Exemplary cleavage-based assay reagents are described in,e.g., Lyamichev et al. (Nat. Biotechnol. 17:292-296, 1999), Ryan et al.(Mol. Diagn. 4:135-144, 1999), and Allawi et al. (J. Clin. Microbiol.44:3443-3447, 2006). Appropriate conditions for flap endonucleasereactions are either known or can be readily determined using methodsknown in the art (see, e.g., Kaiser et al., J. Biol. Chem.274:2138-721394, 1999). Exemplary flap endonucleases that may be used inthe method include Thermus aquaticus DNA polymerase I, Thermusthermophilus DNA polymerase I, mammalian FEN-1, Archaeoglobus fulgidusFEN-1, Methanococcus jannaschii FEN-1, Pyrococcus furiosus FEN-1,Methanobacterium thermoautotrophicum FEN-1, Thermus thermophilus FEN-1,CLEAVASE® (Hologic, Inc., Madison, Wis.), S. cerevisiae RTH1, S.cerevisiae RAD27, Schizosaccharomyces pombe rad2, bacteriophage T5 5′-3′exonuclease, Pyrococcus horikoshii FEN-1, human endonuclease 1, calfthymus 5′-3′ exonuclease, including homologs thereof in eubacteria,eukaryotes, and archaea, such as members of the class II family ofstructure-specific enzymes, as well as enzymatically active mutants orvariants thereof. Descriptions of flap endonucleases can be found in,for example, Lyamichev et al., Science 260:778-783, 1993; Eis et al.,Nat. Biotechnol. 19:673-676, 2001; Shen et al., Trends in Bio. Sci.23:171-173, 1998; Kaiser et al., J. Biol. Chem. 274:21387-21394, 1999;Ma et al., J. Biol. Chem. 275:24693-24700, 2000; Allawi et al., J. Mol.Biol. 328:537-554, 2003; Sharma et al., J. Biol. Chem. 278:23487-23496,2003; and Feng et al., Nat. Struct. Mol. Biol. 11:450-456, 2004.

In certain variations, a cleavage-based assay detects an RNA targetnucleic acid of Lactobacillus sp., G. vaginalis, and/or Eggerthella sp.,and the cleavage-based assay utilizes a flap endonuclease that iscapable of cleaving and RNA:DNA linear duplex structure. In somealternative embodiments, a cleavage-based assay detects a DNA targetnucleic acid of Lactobacillus sp., G. vaginalis, and/or Eggerthella sp.,and the cleavage-based assay utilizes a flap endonuclease that iscapable of cleaving and DNA:DNA linear duplex structure. Exemplary flapendonucleases capable of cleaving RNA:DNA duplexes includepolymerase-deficient 5′ nucleases of the genus Thermus as well ascertain CLEAVASE® enzymes (Hologic, Inc., Madison, Wis.) such as, forexample, CLEAVASE® BN (BstX-NotI deletion of Taq polymerase, see U.S.Pat. No. 5,614,402), CLEAVASE® II (“AG” mutant of full length Taqpolymerase, see U.S. Pat. No. 5,614, 402), CLEAVASE® VII(synthesis-deficient mutation of full length Thermus thermophiluspolymerase), CLEAVASE® IX (polymerase deficient mutant of the Tth DNApolymerase), and CLEAVASE® XII (polymerase deficient chimeric polymeraseconstructed from fragments of taq DNA polymerase and Tth DNApolymerase). Exemplary flap endonucleases capable of cleaving DNA:DNAduplexes include the flap endonucleases indicated above, as well asCLEAVASE® 2.0 (Archaeoglobus fulgidus FEN-1), CLEAVASE® 2.1(Archaeoglobus fulgidus FEN-1 with 6 histidines on the C-terminus),CLEAVASE® 3.0 (Archaeoglobus veneficus FEN-1), and CLEAVASE® 3.1(Archaeoglobus veneficus FEN-1 with 6 histidines on the C-terminus).

In some embodiments, a cleavage-based assay detects an RNA targetnucleic acid of Lactobacillus sp., G. vaginalis, and/or Eggerthella sp.,and the assay includes a step for synthesizing a DNA complement of anRNA target region, which cDNA strand is then hybridized to overlappingfirst and second probe oligonucleotides to form a linear duplex cleavagestructure for cleavage by the flap endonuclease. Reaction conditions forsynthesizing cDNA from an RNA template, using an RNA-dependent DNApolymerase (reverse transcriptase), are well-known in the art.

In certain embodiments utilizing a nucleic-acid-based detection assay,the method further includes purifying the Lactobacillus sp., G.vaginalis, and/or Eggerthella sp. target nucleic acid from othercomponents in the sample. Such purification may include may includemethods of separating and/or concentrating organisms contained in asample from other sample components. In particular embodiments,purifying the target nucleic acid includes capturing the target nucleicacid to specifically or non-specifically separate the target nucleicacid from other sample components. Non-specific target capture methodsmay involve selective precipitation of nucleic acids from asubstantially aqueous mixture, adherence of nucleic acids to a supportthat is washed to remove other sample components, or other means ofphysically separating nucleic acids from a mixture that containsLactobacillus sp., G. vaginalis, and/or Eggerthella sp. nucleic acid andother sample components.

In some embodiments, a target nucleic acid (e.g., a 16S rRNA targetnucleic or a gene encoding the 16S rRNA) of Lactobacillus sp., G.vaginalis, and/or Eggerthella sp. is separated from other samplecomponents by hybridizing the target nucleic acid to a capture probeoligomer. The capture probe oligomer comprises a target-hybridizingsequence configured to specifically or non-specifically hybridize to atarget nucleic acid so as to form a [target nucleic acid]:[captureprobe] complex that is separated from other sample components. Captureprobes comprising target-hybridizing sequences suitable for non-specificcapture of target nucleic acids are described in, e.g., InternationalPCT Publication WO 2008/016988, incorporated by reference herein. Insome specific variations comprising target-hybridizing sequence(s)configured to specifically hybridize to a Lactobacillus sp., G.vaginalis, and/or Eggerthella sp. 16S rRNA target nucleic acid, aLactobacillus-specific capture probe comprises a target-hybridizingsequence substantially corresponding to the nucleotide sequence ofresidues 1-12 of SEQ ID NO:5 (e.g., a capture probe the comprises thetarget-hybridizing sequence of residues 1-12 of SEQ ID NO:5); a G.vaginalis-specific capture probe comprises a target-hybridizing sequencesubstantially corresponding to the nucleotide sequence of residues 1-17of SEQ ID NO:11 (e.g., a capture probe the comprises thetarget-hybridizing sequence of residues 1-17 of SEQ ID NO:11); and/or anEggerthella-specific capture probe comprises a target-hybridizingsequence substantially corresponding to the nucleotide sequence ofresidues 1-21 of SEQ ID NO:15 (e.g., a capture probe the comprises thetarget-hybridizing sequence of residues 1-21 of SEQ ID NO:15). In apreferred variation, the capture probe binds the [target nucleicacid]:[capture probe] complex to an immobilized probe to form a [targetnucleic acid]:[capture probe]:[immobilized probe] complex that isseparated from the sample and, optionally, washed to remove non-targetsample components (see, e.g., U.S. Pat. Nos. 6,110,678; 6,280,952; and6,534,273; each incorporated by reference herein). In such variations,the capture probe oligomer further comprises a sequence or moiety thatbinds attaches the capture probe, with its bound target sequence, to animmobilized probe attached to a solid support, thereby permitting thehybridized target nucleic acid to be separated from other samplecomponents.

In more specific embodiments, the capture probe oligomer includes a tailportion (e.g., a 3′ tail) that is not complementary to target nucleicacid but that specifically hybridizes to a sequence on the immobilizedprobe, thereby serving as the moiety allowing the target nucleic acid tobe separated from other sample components, such as previously describedin, e.g., U.S. Pat. No. 6,110,678, incorporated herein by reference. Anysequence may be used in a tail region, which is generally about 5 to 50nt long, and preferred embodiments include a substantially homopolymerictail of about 10 to 40 nt (e.g., A₁₀ to A₄₀), more preferably about 14to 33 nt (e.g., A₁₄ to A₃₀ or T₃A₁₄ to T₃A₃₀), that bind to acomplementary immobilized sequence (e.g., poly-T) attached to a solidsupport, e.g., a matrix or particle. In some such embodiments comprisingtarget-hybridizing sequence(s) configured to specifically hybridize to aLactobacillus sp., G. vaginalis, and/or Eggerthella sp. 16S rRNA targetnucleic acid, a Lactobacillus-specific capture probe comprises orconsists of a the nucleotide sequence of SEQ ID NO:5; a G.vaginalis-specific capture probe comprises or consists of the nucleotidesequence of SEQ ID NO:11; and/or an Eggerthella-specific capture probecomprises or consists of the nucleotide sequence of SEQ ID NO:15.

Target capture typically occurs in a solution phase mixture thatcontains one or more capture probe oligomers that hybridize to thetarget nucleic acid under hybridizing conditions, usually at atemperature higher than the T_(m) of the [tail sequence]:[immobilizedprobe sequence] duplex. For embodiments comprising a capture probe tail,the [target nucleic acid]:[capture probe] complex is captured byadjusting the hybridization conditions so that the capture probe tailhybridizes to the immobilized probe, and the entire complex on the solidsupport is then separated from other sample components. The support withthe attached [immobilized probe]:[capture probe]:[target nucleic acid]may be washed one or more times to further remove other samplecomponents. Preferred embodiments use a particulate solid support, suchas paramagnetic beads, so that particles with the attached [targetnucleic acid]:[capture probe]:[immobilized probe] complex may besuspended in a washing solution and retrieved from the washing solution,preferably by using magnetic attraction. In embodiments of the methodcomprising the use of an amplification-based detection assay, to limitthe number of handling steps, a target nucleic acid may be amplified bysimply mixing the target nucleic acid in the complex on the support withamplification oligomers and proceeding with amplification steps.

In some embodiments of a method for diagnosing BV, where detection ofLactobacillus sp., G. vaginalis, and/or Eggerthella sp. indicate BV in asubject, the method further includes treating BV in the subject.Treatment regimes for BV are generally known in the art and include, forexample, administration of antibiotic drugs such as metronidazole (e.g.,FLAGYL, METROGEL-VAGINAL), clindamycin (e.g., CLEOCIN, CLINDESSE), andtinidazole (e.g., TINDAMAX). In certain variations, the subject has notbeen previously diagnosed with BV. In other embodiments, the subject hasbeen previously diagnosed with BV and is undergoing treatment for BV atthe time a diagnostic method of the present disclosure is performed.Such variations are particularly useful for monitoring treatment of BVin a subject. For example, if the method indicates that BV is stillpresent in the subject, then the subject may continue treatment. In someembodiments, the same treatment regime (i.e., the same treatment thatthe subject is undergoing at the time the present diagnostic method isperformed) is re-administered to the subject. Alternatively, thecontinued presence of BV in the subject undergoing treatment mayindicate that a change in the ongoing treatment is needed, and adifferent treatment regime (e.g., a different medication, or anincreased dosage and/or frequency of a drug) is administered to thesubject.

In accordance with the present invention, detecting the presence orabsence of Lactobacillus sp., G. vaginalis, and/or Eggerthella sp. maybe performed separately for each target (e.g., in separate reactionvessels, sequentially or in parallel), or performed together as amultiplex reaction system. Accordingly, in some embodiments, a method asdescribed herein (e.g., a method for diagnosing BV) utilizes a multiplexreaction, where the reaction mix contains reagents for assaying multiple(e.g., at least two, three, four, or more) different target sequences inparallel. In these cases, a reaction mix may contain multiple differenttarget-specific oligonucleotides for performing the detection assay. Forexample, in a method utilizing an amplification-based detection assay, amultiplex reaction may contain multiple sets (e.g., multiple pairs) ofamplification oligomers (for example, multiple pairs of PCR primers ormultiple pairs of TMA amplification oligomers (e.g., for TMA, multiplepairs of promoter primer and non-promoter primer, or multiple pairs ofpromoter provider and non-promoter primer)). In other embodimentsutilizing a cleavage-based detection assay, a multiplex reaction maycontain multiple probe oligonucleotides having different flaps, multipledifferent overlapping probe oligonucleotides, and multiple differentFRET cassettes for detecting the different flaps, once they are cleaved.

Additional microbe detection assays can be similarly performed fordetermining the presence and/or relative amount of a plurality ofmicrobes implicated in BV. By way of example only, such plurality ofmicrobes can include one or more of anaerobic gram-positive cocci;Trichomonas sp.; Trichomonas vaginalis; Candida sp.; Eggerthella sp.;Bacterium from the order Clostridiales; Clostridium-like sp.; Atopobiumsp.; Atopobium vaginae; Enterobacteria; Peptostreptococcus micros;Aerococcus christensenii; Leptotrichia amnionii; Peptoniphilus sp.;Dialister sp.; Mycoplasma hominis; Sneathia sanguinegens; Anaerococcustetradius; Mobiluncus sp.; Mobiluncus hominis; Eggerthellahongkongensis; Prevotella sp.; Megasphaera sp.; Leptotrichiasanguinegens and Finegoldia magna. Assays may be performed separately ormultiplexed. Thus, a diagnosis of BV can include identifying a pluralityof microbes and optionally determining their relative abundances in asample.

In certain embodiments, the method for diagnosing BV includes thedetection of no more than ten bacterial genera associated with BV. Inother embodiments, the method includes the detection of no more thannine, no more than eight, no more than seven, no more than six, no morethan five, or nor more than four bacterial genera associated with BV. Insome variations, the method does not include detection of bacterialgenera associated with BV other than Lactobacillus, Gardnerella, andEggerthella.

Also provided by the subject invention is an oligomer for determiningthe presence or absence of any one or more of Lactobacillus sp., G.vaginalis, and Eggerthella sp. in a sample. In various embodiments, theoligomer combination includes oligomers as set forth herein for a methodfor determining the presence or absence of Lactobacillus sp., G.vaginalis, and/or Eggerthella sp. in a sample. In some variations, theoligomer combination includes at least one Lactobacillus-specificoligonucleotide (e.g., at least two or three Lactobacillus-specificoligonucleotides, each binding to different target sequences); at leastone G. vaginalis-specific oligonucleotide (e.g., at least two or threeG. vaginalis-specific oligonucleotides, each binding to different targetsequences); and at least one Eggerthella-specific oligonucleotide (e.g.,at least two or three Eggerthella-specific oligonucleotides, eachbinding to different target sequences). In some variations, the oligomercombination includes at least two Lactobacillus-specificoligonucleotides (e.g., at least three Lactobacillus-specificoligonucleotides, each binding to different target sequences); at leasttwo G. vaginalis-specific oligonucleotides (e.g., at least three G.vaginalis-specific oligonucleotides, each binding to different targetsequences); and/or at least two Eggerthella-specific oligonucleotides(e.g., at least three Eggerthella-specific oligonucleotides, eachbinding to different target sequences). In some embodiments, theoligomer combination at least two Lactobacillus-specific amplificationoligonucleotides for amplifying a Lactobacillus sp. nucleic acid targetregion; at least two G. vaginalis-specific amplificationoligonucleotides for amplifying a G. vaginalis nucleic acid targetregion; and/or at least two Eggerthella-specific amplificationoligonucleotides for amplifying an Eggerthella sp. nucleic acid targetregion. The oligomer combination may be in the form of a reactionmixture or a kit comprising the oligomers. The reaction mixture or kitmay further include a number of optional components such as, forexample, capture probe nucleic acids or arrays of capture probe nucleicacids. For an amplification reaction mixture, the reaction mixture willtypically include other reagents suitable for performing in vitroamplification such as, e.g., buffers, salt solutions, appropriatenucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTPand UTP), and/or enzymes (e.g., reverse transcriptase, and/or RNApolymerase), and will typically include test sample components, in whichan Eggerthella, Prevotella, and/or Lactobacillus target nucleic acid mayor may not be present. A kit comprising an oligomer combination foramplification of one or more target nucleic acid regions ofLactobacillus sp., G. vaginalis, and/or Eggerthella sp. may also includeother reagents suitable for performing in vitro amplification such as,e.g., buffers, salt solutions, appropriate nucleotide triphosphates(e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP and UTP), and/or enzymes(e.g., reverse transcriptase, and/or RNA polymerase). For an oligomercombination (e.g., reaction mixture or kit) that includes a detectionprobe together with an amplification oligomer combination targeting acommon target nucleic acid, selection of amplification oligomers anddetection probe oligomers are linked by a common target region (i.e.,the combination will include a probe that binds to a sequenceamplifiable by the amplification oligomer combination).

The invention is further illustrated by the following non-limitingexamples.

EXAMPLE 1 Reagents for RT-TMA-Based Assays

Unless otherwise specified, reagents commonly used in the RT-TMA-basedassays described herein include the following. Target Capture Reagent(TCR), General Purpose Reagent formulation: 250 mM HEPES, 1.88 M LiCl,310 mM LiOH, 100 mM EDTA, pH 6.4, and 250 μg/ml of paramagneticparticles (0.7-1.05 micron particles, Sera-Mag™ MG-CM) with (dT)₁₄oligomers covalently bound thereto. Wash Solution: 10 mM HEPES, 150 mMNaCl, 6.5 mM NaOH, 1 mM EDTA, 0.3% (v/v) ethanol, 0.02% (w/v)methylparaben, 0.01% (w/v) propylparaben, and 0.1% (w/v) sodium laurylsulfate, pH 7.5. Amplification reagent, Real-Time Reagent formulation: asolution containing 11.61 mM Tris base, 14.94 mM Tris-HCl, 28.5 mMMgCl₂, 23.30 mM KCl, 3.3% Glycerol, 0.02% PRO CLIN 300, 0.05 mM ZincAcetate Dihydrate, 0.76 mM each of dATP, dCTP, dGTP, and dTTP, 6.50 mMeach ATP, CTP, and GTP, 7.50 mM UTP, to which primers may be added.RT-TMA Enzymes: 57.46 mM HEPES, 49.58 mM N-Acetyl-L-Cysteine, 0.98 mMEDTA free acid, 0.039 mM EDTA Disodium Dihydrate, 0.10 v/v TRITON X-100,49.61 mM KCl, 0.20 v/v Glycerol, 0.03 w/v Trehalose Dihydrate, MMLVreverse transcriptase (RT) and T7 RNA polymerase. Promoter Reagent,Real-Time Reagent formulation: same reagent formulation as AmplificationReagent.

Oligo screening experiments for amplification oligos and torches wereperformed on an OEM platform (Stratagene Mx3000) using the biphasicreal-time TMA format. Briefly, samples were incubated with 100 μl TCRreagent, containing target-specific capture oligos (15 pmol/rxn) and T7oligos (5 pmol/rxn) at 62° C. for 30 minutes, then ramped down to roomtemperature for 20 minutes. Magnetic beads bound with TCOs and targetrRNAs were washed and eluted into the Amplification reagent, containing15 pmol/rxn NT7 oligos. Samples were incubated at 43° C. during additionof Enzyme Reagent (25 μl) and subsequent addition of Promoter Reagent(25 μl). The Promoter Reagent contained 15 pmol/rxn T7 oligos and 15pmol/rxn Torch oligos. Fluorescence emission, reflecting Torch bindingto target amplicon rRNA and resulting in dye separation from quencher,was measured in real-time on the Stratagene instrument every 30 secondsfor 1 hour. Fluorescence curve profiles were analyzed for amplificationof target.

Several T7 oligos were screened for the Lactobacillus sp. targetsystems, to identify the optimal combination of these oligos for overallL. spp target detection in the assay. Two T7 oligos were selected forinclusion in the assay. Additional torch oligos were developed andtested to improve the detection of L. gasseri specifically, and reducecross-detection of Lactobacillus iners (a non-target Lactobacillusspecies). Several torches showed good performance for L. gasseri, andone showed the best reduction in L. iners cross-detection. Two differentT7 oligos for G. vaginalis were screened for possible inclusion of acompetitive internal control for the assay, utilizing the T7/NT7 oligosfor G. vaginalis and torch-specific oligos for G. vaginalis versus ICdetection. Different target-specific target capture oligos were designedand screened, comparing performance of the assay for each target systemto a common BV target capture oligo. One target-specific TCO wasselected for each BV target system. Additional TCOs were screened forthe L. spp targets, and one TCO was selected that provided the bestbalance in detection for the three L. spp targets overall. A list of theprimers screened for optimization of the multiplex bacterial vaginosisassay are listed in Table 1.

Following optimization of target amplification and detection, oligoswere selected for each BV target and subsequent experiments were carriedout on the Panther instrument platform. The amplification and detectionreaction was configured to amplify and detect all specified BV targetsas well as a nucleic acid internal control. Briefly, lysed target wascombined with target capture oligos, magnetic beads joined toimmobilized probes, and T7 primers. Reaction conditions were provided tohybridize the target capture oligos and the T7 primers to their intendedtargets. A series of wash steps were performed to remove cellularcomponents, culture and transport medium and the like. Following thewash step, a first amplification reagent was added to the washed andcaptured taregt nucleic acids. The first amplification reagent containedonly non-T7 primers. There was no primer-annealing step prior to enzymeaddition; initiation of amplification occured at addition of enzyme, andwas followed by a 5-minute incubation step at 42° C. The secondamplification reagent containing T7 primers and torches as detectionprobes was added to the first amplification reagent after a 5-minute 42°C. incubation reaction. Real-time detection occurred during this stepmeasured by 4 separate fluorometers used to detect Fam, Hex, Rox, andCy5.5 dye labels on each of the different detection probes.

EXAMPLE 2 Sensitivity and Specificity Testing of Oligomer Combinationsfor Amplification and Detection of BV Target Nucleic Acid

Analytical sensitivity of BV targets was assessed testing half-logtitrations of lysates (for L. spp targets and G. vaginalis), or in vitrotranscripts (IVTs) for Eggerthella, spiked into Aptima SpecimenTransport Medium (STM). Fifteen replicates were screened at each panelconcentration (N=10 total concentrations, for each target) on thePanther instrument. Oligos were tested in multiplex-kit format; however,each BV target was screened separately. The assays used TCO primers (15pmol/rxn), T7 primers (at either 5 or 15 pmol/rxn), NT7 primers (15pmol/rxn), and Torch primers (15 pmol/rxn). A Probit analysis wasperformed to determine the 95% and 50% detection levels for each of theBV targets. At 95% Probability (95% CL), limit-of-detection (LoD) wasdetermined: L. crispatus, 3.4e4 (3.2e4-3.5e4) CFU/ml; L. jensenii, 3.1e3(3.0e3-3.2e3) CFU/ml; L. gasseri, 3.2e3 (3.1e3-3.2e3) CFU/ml; G.vaginalis, 107 (103-110) CFU/ml; Eggerthella, 1.1e8 (1.0e8-1.2e8)copies/ml.

Cross-reactivity of optimal primer sets for each BV target was testedagainst 16 pooled panels of 4-5 organisms each. No significantcross-detection of any of the organisms was observed for the primer setoptimized to detect G. vaginalis. The primer set optimized to detect theL. spp targets cross-reacted with 1 of the pooled panels screened.Additional experiments determined that the cross-reacting organism wasLactobacillus acidophilus. L. acidophilus is a gut microbe and wasdetermined to be no risk for the assay. The primer set optimized todetect the Eggerthella target cross-reacted with 1 of the pooled panelsscreened. Additional experiments determined that the cross-reactingorganism was Neisseria gonorrhoeae. N. gonorrhoeae was a high-riskcross-detection, and additional primers were screened for Eggerthella.The new optimized primer set successfully eliminated detection of N.gonorrhoeae up to 1e7 CFU/ml.

EXAMPLE 3 Clinical Sensitivity of RT-TMA Assay for Detecting BV TargetNucleic Acids

Clinical performance of the bacterial vaginosis assay was assessed usingvaginal swab patient specimens (n=200, collected from four differentclinical sites), and included roughly 100 positive and 100 negativesamples (utilizing Cartwright criteria). TTime was used as a cutoff ineach fluorescent channel utilized for target detection (Fam, Hex,Cy5.5): L. spp, 25 minutes; G. vaginalis, 12.5 minutes; Eggerthella, 40minutes. The bacterial vaginosis assay clinical performance was analyzedfor sensitivity: 93.8% (86.2-97.3), and specificity: 88.6% (79.7-93.9).

Ninety-eight ThinPrep/vaginal swab paired samples were also assessed forclinical feasibility and the bacterial vaginosis assay performance,including a roughly equal amount of positive and negative samples(utilizing Cartwright criteria). Overall agreement for positivesamples=93.8%; overall agreement for negative samples=100%; overallagreement for intermediate samples=87.5%.

EXAMPLE 4 Clinical Sensitivity of RT-TMA Assay for Detecting BV TargetNucleic Acids

For this testing, Amplification and Promoter reagent formulations wereadjusted compared to those previously described in Example 1.Amplification reagent, Real-Time Reagent formulation: a solutioncontaining 11.61 mM Tris base, 14.94 mM Tris-HCl, 31.0 mM MgCl₂, 23.30mM KCl, 3.3% Glycerol, 0.02% PRO CLIN 300, 0.05 mM Zinc AcetateDihydrate, 0.83 mM each of dATP, dCTP, dGTP, and dTTP, 7.00 mM each ATP,CTP, and GTP, 8.00 mM UTP, to which primers may be added. RT-TMAEnzymes: 57.46 mM HEPES, 49.58 mM N-Acetyl-L-Cysteine, 0.98 mM EDTA freeacid, 0.039 mM EDTA Disodium Dihydrate, 0.10 v/v TRITON X-100, 49.61 mMKCl, 0.20 v/v Glycerol, 0.03 w/v Trehalose Dihydrate, MMLV reversetranscriptase (RT) and T7 RNA polymerase. Promoter Reagent, Real-TimeReagent formulation: same reagent formulation as Amplification Reagent.

Oligo concentration optimization experiments were carried out todetermine the optimal concentration for each oligo of the bacterialvaginosis assay. The oligo concentrations were titrated and screenedagainst target IVTs spiked into STM at concentrations that woulddemonstrate differences in performance. From this testing it wasdetermined to adjust the oligo concentrations to optimize performance ofthe assay. For the TCR reagent, the TCOs for Lactobacillus sp.,Eggerthella, and Gardnerella were adjusted from 15 pmol/rxn to 10pmol/rxn. All T7 primers were removed from the TCR reagent. For theAmplification reagent, the NT7 for Eggerthella was adjusted from15pmol/rxn to 10 pmol/rxn, and the NT7s for Lactobacillus andGardnerella were adjusted from 15pmol/rxn to 5 pmol/rxn. For the T7oligos in the Promoter reagent, Lactobacillus sp. T7 oligo SEQ ID NO:7was removed from the formulation, Lactobacillus T7 oligo SEQ ID NO:8remained in the formulation and was adjusted from 15 pmol/rxn to 4pmol/rxn, the Eggerthella T7 was adjusted from 15 pmol/rxn to 10pmol/rxn, and the Gardnerella T7 was adjusted from 15 pmol/rxn to 5pmol/rxn. For the torch oligos in the Promoter reagent, theLactobacillus sp. torch for L. gasseri detection (SEQ ID NO:10) wasadjusted from 15 pmol/rxn to 10 pmol/rxn, the Lactobacillus sp. torchfor L. crispatus and L. jensenii detection was adjusted from 15 pmol/rxnto 20 pmol/rxn, and the Gardnerella torch and Eggerthella torch wereadjusted from 15 pmol/rxn to 20 pmol/rxn.

Clinical performance of the bacterial vaginosis assay was assessed usingvaginal swab patient specimens (n=353, collected from three differentclinical sites), and included 78 positive, 41 intermediates, and 234negatives according to culture result. Intermediate status was resolvedwith Amsel criteria resulting in 4 positive and 37 negative. Positivecriteria was determined if the sample had an RFU signal that exceeded2000 RFU in FAM, 2000 RFU in HEX, and/or 1600 RFU in ROX. A positiveresult in FAM channel corresponded to positive result for Lactobacillussp., a positive result in HEX corresponded to a positive result forEggerthella, and a positive result in the ROX channel corresponded to apositive all for Gardnerella. No TTime cutoff was utilized to determinea positive result. The bacterial vaginosis assay clinical performancewas analyzed for sensitivity and specificity with and without theinclusion of the Eggerthella target. The sensitivity of the assay was96.3 (89.7-99.2) with Eggerthella and 96.3 (89.7-99.2) withoutEggerthella. The specificity of the assay was 86.7 (82.1-90.5) withEggerthella and 87.5 (82.9-91.2) without Eggerthella. Better specificityis gained with the exclusion of Eggerthella detection from the sampletesting results (the improved specificity was due to removal of falsepositives). Furthermore, the Eggerthella result was only found to bepositive when Gardnerella was also determined to be positive a result,and no BV positive sample was determined by using Eggerthella statusalone. The results demonstrate that improved specificity is gained bythe exclusion of Eggerthella from the BV assay formulation and thussupports the removal from the assay.

TABLE 3 Oligos Screened for the Bacterial Vaginosis Assay SEQ ID NO:Sequence (5′ → 3′) Type 12 CTTACCTGGGCTTGACATGTGCCTG NT7 19GGATTCATTGGGCGTAAAGC NT7  6 CGGATGGGTGAGTAAC NT7 20 GCGTTATCCGGATTCATNT7 16 AGCGTTATCCGGATTC NT7 13 AATTTAATACGACTCACTATAGGGAGACACCACCTGTGAACT7 CTGC 17 AATTTAATACGACTCACTATAGGGAGATTCGGAACCCGGCT T7 CGAGGTTAAG 21AATTTAATACGACTCACTATAGGGAGATAAGCCTTTACCTT T7 ACCA 22AATTTAATACGACTCACTATAGGGAGAATACGACAGCTTAC T7 GCCGC 23AATTTAATACGACTCACTATAGGGAGATATCTGCGCATTTC T7 ACCGCTACAC  8AATTTAATACGACTCACTATAGGGAGATAAGCCGTTACCTT T7 ACCA  7AATTTAATACGACTCACTATAGGGAGATAAGCCCTTACCTT T7 ACCA 24AATTTAATACGACTCACTATAGGGAGACGACCATGCACCAC T7 CTGT 25GGACUACCAGGGUAUCUAAUCCUGTTTAAAAAAAAAAAAA TCO AAAAAAAAAAAAAAAAA 11CAUGCUCCGCCGCUUGUTTTAAAAAAAAAAAAAAAAAAAA TCO AAAAAAAAAA 15GUACCGUCGAUGUCUUCCCUGTTTAAAAAAAAAAAAAAAA TCO AAAAAAAAAAAAAA 26GUAGGAGUUUGGGCCGUGUTTTAAAAAAAAAAAAAAAAAA TCO AAAAAAAAAAAA  5UCUGUUAGUUCCTTTAAAAAAAAAAAAAAAAAAAAAAAAA TCO AAAAA 27CUUUGAGUUUUAGCCUUGCGTTTAAAAAAAAAAAAAAAAA TCO AAAAAAAAAAAAA 28GCAUCGAAUUAAUCCGCAUGCTTTAAAAAAAAAAAAAAAA TCO AAAAAAAAAAAAAA 29GUAGUUAGCCGGGGCUUCUUCTTTAAAAAAAAAAAAAAAA TCO AAAAAAAAAAAAAA 30UACGUAUUACCGCGGCUGTTTAAAAAAAAAAAAAAAAAAA TCO AAAAAAAAAAA 31CGUGUCUCAGUCCCAAUGUGTTTAAAAAAAAAAAAAAAAA TCO AAAAAAAAAAAAA 32CAAUGUGGCCGAUCAGUCTTTAAAAAAAAAAAAAAAAAAA TCO AAAAAAAAAAA 33CCAUUGUGGAAGAUUCCCUACTTTAAAAAAAAAAAAAAAA TCO AAAAAAAAAAAAAA 34CUCGCUCGACUUGCAUGUATTTAAAAAAAAAAAAAAAAAA TCO AAAAAAAAAAAA 35CUUGCAUGUAUUAGGCACGTTTAAAAAAAAAAAAAAAAAA TCO AAAAAAAAAAAA 36CUUGUAUCUAUGUCCAUUCCTTTAAAAAAAAAAAAAAAAA TCO AAAAAAAAAAAAA 37UGGUGCAAGCACCAAAUUCTTTAAAAAAAAAAAAAAAAAA TCO AAAAAAAAAAAA 38AAUUCAUCUAGGCAAGTTTAAAAAAAAAAAAAAAAAAAAA TCO AAAAAAAAA 39UCCUAACGUCAUUACCTTTAAAAAAAAAAAAAAAAAAAAA TCO AAAAAAAAA 40UCCAUUCCGAAGAATTTAAAAAAAAAAAAAAAAAAAAAAA TCO AAAAAAA 41UUGCUGCGUCAGGGUUUCCTTTAAAAAAAAAAAAAAAAAA TCO AAAAAAAAAAAA 42GAAAGCGGUUUACAACCCGATTTAAAAAAAAAAAAAAAAA TCO AAAAAAAAAAAAA 43AAGGCCUUCAUCCCGCATTTAAAAAAAAAAAAAAAAAAAA TCO AAAAAAAAAA 44UCGCCGUUGGUGUUCUUCTTTAAAAAAAAAAAAAAAAAAA TCO AAAAAAAAAAA 45UGACGGCCCAGCAGACUTTTAAAAAAAAAAAAAAAAAAAA TCO AAAAAAAAAA 46UCCUGUUCGCUCCCCCAGCUTTTAAAAAAAAAAAAAAAAA TCO AAAAAAAAAAAAA 47TCCTAACGTCATTACCTTTAAAAAAAAAAAAAAAAAAAAAA TCO AAAAAAAA 48GTACCGTCGATGTCTTCCCTGTTTAAAAAAAAAAAAAAAAA TCO AAAAAAAAAAAAA 49CAGCGCUCAUCGUUTTTAAAAAAAAAAAAAAAAAAAAAAA TCO AAAAAAA 50ACGGAAGAUGUAAUCUCTTTAAAAAAAAAAAAAAAAAAAA TCO AAAAAAAAAA 51GAUGUAAUCUCCCACTTTAAAAAAAAAAAAAAAAAAAAAA TCO AAAAAAAA 52CCAUUCCGAAGATTTAAAAAAAAAAAAAAAAAAAAAAAAA TCO AAAAA 53AGUUUCAUCUGTTTAAAAAAAAAAAAAAAAAAAAAAAAAA TCO AAAA 54CCGAAGAAUUUCTTTAAAAAAAAAAAAAAAAAAAAAAAAA TCO AAAAA 55UUCAAUAGGCTTTAAAAAAAAAAAAAAAAAAAAAAAAAAA TCO AAA 56CGUCAUUACCTTTAAAAAAAAAAAAAAAAAAAAAAAAAAA TCO AAA 57CCAAAUUCAUCUTTTAAAAAAAAAAAAAAAAAAAAAAAAA TCO AAAAA 58AACGUCAUUACCTTTAAAAAAAAAAAAAAAAAAAAAAAAA TCO AAAAA 59UUUCCUAACGUCTTTAAAAAAAAAAAAAAAAAAAAAAAAA TCO AAAAA 60CCUGCAGAGAUGUGGUUUCGCAGG Torch 18 CCGCUCAGGCGGUUGCUCAAGCGG Torch  9GUCUGGGAUACCACUUGGAAACAGAC Torch 61 CUGGGAUACCACUUGGAAACACCCAG Torch 62UCUGGGAUACCACUUCCAGA Torch 14 CCUGCAGAGAUGUGGUUUCGCAGG Torch 63CCUGCAGAGAUGUGGUUUCGCAGG Torch 64 GGUUCUGCUAUCACUCUUGGAACC Torch 65ACGCAUGUCUAGAGUUGCGU Torch 66 AGACGCAUGUCUAGAGCGUCU Torch 67GAUGCUAAUACCGGAUAACAACGCAUC Torch 68 UACCGGAUAACAACACUAGACGCCGGUA Torch69 GUCUGGGAUACCAUUUGGAAACAGAC Torch 70 GUCUGGGAUACCACUUGGAAACAGAC Torch71 GUCUGGGAUACCACUUGGAAACAGAC Torch 72 GGUUCUGCUAUCACUCUUGGAACC Torch 73GGUUCUGCUAUCACUCUUGGAACC Torch 74 CGCAUGUCUAGAGUUGCG Torch 75ACUCACGCAUGUCUAGAGU Torch 10 CACUCACGCAUGUCUAGAGUG Torch 76GAGACGCAUGUCUAGAGUUGUCUC Torch 77 GUUGCUCAAGCGGAACCUCGCAAC Torch 78UUGCUCAAGCGGAACCUCUAGCAA Torch 79 GCUCAAGCGGAACCUCUGAGC Torch 80AGCGGAACCUCUAAUCUCGUCGCU Torch 81 CCGCUCAGGCGGUUGCUCAAGCGG Torch 82CCGCUCAGGCGGUUGCUCAAGCGG Torch 83 CCGCUCAGGCGGUUGCUCAAGCGG Torch 84CCGCUCAGGCGGUUGCUCAAGCGG Torch 85 CCGCUCAGGCGGUUGCUCAAGCGG Torch 86CCGCUCAGGCGGUUGCUCAAGCGG Torch 87 CCGCUCAGGCGGUUGCUCAAGCGG Torch

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims. All publications, patents, andpatent applications cited herein are hereby incorporated by reference intheir entireties for all purposes.

What is claimed is:
 1. A multiplex method for determining the presenceor absence of each of Lactobacillus sp., G. vaginalis, and Eggerthellasp. in a sample, the method comprising: (1) contacting a sample, saidsample suspected of containing at least one of Lactobacillus sp., G.vaginalis, and Eggerthella sp., with (a) first and secondLactobacillus-specific amplification oligomers for amplifying a targetregion of a Lactobacillus sp. target nucleic acid, wherein (i) the firstLactobacillus-specific amplification oligomer comprises a firstLactobacillus-specific target-hybridizing sequence substantiallycorresponding to the nucleotide sequence of residues 28-45 of SEQ IDNO:7 or residues 28-45 of SEQ ID NO:8 and (ii) the secondLactobacillus-specific amplification oligomer comprises a secondLactobacillus-specific target-hybridizing sequence substantiallycorresponding to the nucleotide sequence of SEQ ID NO:6; (b) first andsecond G. vaginalis-specific amplification oligomers for amplifying atarget region of a G. vaginalis target nucleic acid, wherein (i) thefirst G. vaginalis-specific amplification oligomer comprises a first G.vaginalis-specific target-hybridizing sequence substantiallycorresponding to the nucleotide sequence of residues 28-45 of SEQ IDNO:13 and (ii) the second G. vaginalis-specific amplification oligomercomprises a second G. vaginalis-specific target-hybridizing sequencesubstantially corresponding to the nucleotide sequence of SEQ ID NO:12;and (c) first and second Eggerthella-specific amplification oligomersfor amplifying a target region of an Eggerthella sp. target nucleicacid, wherein (i) the first Eggerthella-specific amplification oligomercomprises a first Eggerthella-specific target-hybridizing sequencesubstantially corresponding to the nucleotide sequence of residues 28-51of SEQ ID NO:17 and (ii) the second Eggerthella-specific amplificationoligomer comprises a second Eggerthella-specific target-hybridizingsequence substantially corresponding to the nucleotide sequence of SEQID NO:16; (2) performing an in vitro nucleic acid amplificationreaction, wherein any Lactobacillus sp., G. vaginalis, and Eggerthellasp. target nucleic acid, if present in the sample, is used as a templatefor generating one or more amplification products corresponding to theLactobacillus sp., G. vaginalis, and Eggerthella sp. target regions; and(3) detecting the presence or absence of the one or more amplificationproducts, thereby determining the presence or absence of Lactobacillussp., G. vaginalis, and Eggerthella sp. in the sample.
 2. The method ofclaim 1, wherein the first Lactobacillus-specific target-hybridizingsequence substantially corresponds to the nucleotide sequence ofresidues 28-45 of SEQ ID NO:7, and wherein the sample is furthercontacted with a third Lactobacillus-specific amplification oligomer foramplifying the Lactobacillus sp. target region, wherein the thirdLactobacillus-specific amplification oligomer comprises a thirdLactobacillus-specific target-hybridizing sequence substantiallycorresponding to the nucleotide sequence of residues 28-45 of SEQ IDNO:8.
 3. The method of claim 1, wherein the first Lactobacillus-specifictarget-hybridizing sequence comprises the nucleotide sequence ofresidues 28-45 of SEQ ID NO:7 or residues 28-45 of SEQ ID NO:8; thesecond Lactobacillus-specific target-hybridizing sequence comprises thenucleotide sequence of SEQ ID NO:6; the first G. vaginalis-specifictarget-hybridizing sequence comprises the nucleotide sequence ofresidues 28-45 of SEQ ID NO:13; the second G. vaginalis-specifictarget-hybridizing sequence comprises the nucleotide sequence of SEQ IDNO:12; the first Eggerthella-specific target-hybridizing sequencecomprises the nucleotide sequence of residues 28-51 of SEQ ID NO:17;and/or the second Eggerthella-specific target-hybridizing sequencecomprises the nucleotide sequence of SEQ ID NO:16.
 4. The method ofclaim 1 or 3, wherein the first Lactobacillus-specifictarget-hybridizing sequence substantially corresponds to the nucleotidesequence of residues 28-45 of SEQ ID NO:7, and wherein the sample isfurther contacted with a third Lactobacillus-specific amplificationoligomer for amplifying the Lactobacillus sp. target region, wherein thethird Lactobacillus-specific amplification oligomer comprises a thirdLactobacillus-specific target-hybridizing sequence comprising thenucleotide sequence of residues 28-45 of SEQ ID NO:8.
 5. The method ofclaim 1 or 3, wherein the first Lactobacillus-specifictarget-hybridizing sequence consists of the nucleotide sequence ofresidues 28-45 of SEQ ID NO:7 or residues 28-45 of SEQ ID NO:8; thesecond Lactobacillus-specific target-hybridizing sequence consists ofthe nucleotide sequence of SEQ ID NO:6; the first G. vaginalis-specifictarget-hybridizing sequence consists of the nucleotide sequence ofresidues 28-45 of SEQ ID NO:13; the second G. vaginalis-specifictarget-hybridizing sequence consists of the nucleotide sequence of SEQID NO:12; the first Eggerthella-specific target-hybridizing sequenceconsists of the nucleotide sequence of residues 28-51 of SEQ ID NO:17;and/or the second Eggerthella-specific target-hybridizing sequenceconsists of the nucleotide sequence of SEQ ID NO:16.
 6. The method ofany of claims 1, 3, and 5, wherein the first Lactobacillus-specifictarget-hybridizing sequence substantially corresponds to the nucleotidesequence of residues 28-45 of SEQ ID NO:7, and wherein the sample isfurther contacted with a third Lactobacillus-specific amplificationoligomer for amplifying the Lactobacillus sp. target region, wherein thethird Lactobacillus-specific amplification oligomer comprises a thirdLactobacillus-specific target-hybridizing sequence consisting of thenucleotide sequence of residues 28-45 of SEQ ID NO:8.
 7. The method ofany one of claims 1 to 6, wherein at least one of the firstLactobacillus-specific amplification oligomer, the first G.vaginalis-specific amplification oligomer, and the firstEggerthella-specific amplification oligomer is a promoter primer orpromoter provider further comprising a promoter sequence located 5′ tothe respective target hybridizing sequence.
 8. The method of any one ofclaims 1, 3, 5, and 7, wherein the first Lactobacillus-specifictarget-hybridizing sequence substantially corresponds to the nucleotidesequence of residues 28-45 of SEQ ID NO:7, and wherein the sample isfurther contacted with a third Lactobacillus-specific amplificationoligomer for amplifying the Lactobacillus sp. target region, wherein thethird Lactobacillus-specific amplification oligomer is a promoter primeror promoter provider comprising a third Lactobacillus-specifictarget-hybridizing sequence substantially corresponding to thenucleotide sequence of residues 28-45 of SEQ ID NO:8 and furthercomprising a promoter sequence located 5′ to the thirdLactobacillus-specific target hybridizing sequence.
 9. The method of anyof claims 2, 4, and 6, wherein the third Lactobacillus-specificamplification oligomer is a promoter primer or promoter provider furthercomprising a promoter sequence located 5′ to the thirdLactobacillus-specific target hybridizing sequence.
 10. The method ofclaim 7 or 9, wherein the promoter sequence is a T7 promoter sequence.11. The method of claim 10, wherein the promoter sequence has thenucleotide sequence of residues 1-27 of SEQ ID NO:7.
 12. The method ofclaim 7, wherein the first Lactobacillus-specific amplification oligomerhas the nucleotide sequence of SEQ ID NO:7; the first G.vaginalis-specific amplification oligomer has the nucleotide sequence ofSEQ ID NO:13; and/or the first Eggerthella-specific amplificationoligomer has the nucleotide sequence of SEQ ID NO:17.
 13. The method ofclaim 9, wherein the third Lactobacillus-specific amplification oligomerhas the nucleotide sequence of SEQ ID NO:8.
 14. The method of any one ofclaims 1 to 13, further comprising purifying the Lactobacillus, G.vaginalis, and Eggerthella target nucleic acids, if present, from othercomponents in the sample before step (2).
 15. The method of claim 14,wherein the purifying step comprises contacting the sample with at leastone capture probe oligomer comprising a target-hybridizing sequencecovalently attached to a sequence or moiety that binds to an immobilizedprobe.
 16. The method of claim 15, wherein the sample is contacted witha Lactobacillus-specific capture probe oligomer, a G. vaginalis-specificcapture probe oligomer, and an Eggerthella-specific capture probeoligomer, wherein each of the Lactobacillus-specific, G.vaginalis-specific, and Eggerthella-specific capture probe oligomerscomprises a capture probe target-hybridizing sequence that specificallyhybridizes to a target sequence within the Lactobacillus, G. vaginalis,or Eggerthella target nucleic acid, respectively, and wherein each ofthe Lactobacillus-specific, G. vaginalis-specific, andEggerthella-specific capture probe target-hybridizing sequences iscovalently attached to the sequence or moiety that binds to theimmobilized probe.
 17. The method of claim 16, wherein theLactobacillus-specific capture probe target-hybridizing sequencesubstantially corresponds to the nucleotide sequence of residues 1-12 ofSEQ ID NO:5, the G. vaginalis-specific capture probe target-hybridizingsequence substantially corresponds to the nucleotide sequence ofresidues 1-17 of SEQ ID NO:11, and/or the Eggerthella-specific captureprobe target hybridizing sequence substantially corresponds to thenucleotide sequence of residues 1-21 of SEQ ID NO:15.
 18. The method ofclaim 17, wherein the Lactobacillus-specific capture probe oligomer hasthe nucleotide sequence of SEQ ID NO:5, the G. vaginalis-specificcapture probe oligomer has the nucleotide sequence of SEQ ID NO:11,and/or the Eggerthella-specific capture probe oligomer has thenucleotide sequence of SEQ ID NO:15.
 19. The method of any of claims 1to 18, wherein the detecting step (3) comprises contacting the one ormore amplification products with a first Lactobacillus-specificdetection probe that specifically hybridizes to the Lactobacillus sp.target region, a first G. vaginalis-specific detection probe thatspecifically hybridizes to the G. vaginalis target region, and a firstEggerthella-specific detection probe that specifically hybridizes to theEggerthella sp. target region, and detecting the presence or absence ofany target-hybridized Lactobacillus-specific, G. vaginalis-specific,and/or Eggerthella-specific detection probe.
 20. The method of claim 19,wherein the first Lactobacillus-specific detection probe comprises atarget-hybridizing sequence substantially corresponding to SEQ ID NO:9,the first G. vaginalis-specific detection probe comprises atarget-hybridizing sequence substantially corresponding to residues 1-19of SEQ ID NO:14, and/or the first Eggerthella-specific detection probecomprises a target-hybridizing sequence substantially corresponding toSEQ ID NO:18.
 21. The method of claim 20, wherein the firstLactobacillus-specific detection probe has the nucleotide sequence ofSEQ ID NO:9, the first G. vaginalis-specific detection probe has thenucleotide sequence of SEQ ID NO:14, and/or the firstEggerthella-specific detection probe has the nucleotide sequence of SEQID NO:18.
 22. The method of claim 20 or 21, wherein the detecting step(3) further comprises contacting the one or more amplification productswith a second Lactobacillus-specific detection probe that specificallyhybridizes to the Lactobacillus sp. target region, wherein the secondLactobacillus-specific detection probe comprises a target-hybridizingsequence substantially corresponding to residues 6-21 of SEQ ID NO:10.23. The method of claim 22, wherein the second Lactobacillus-specificdetection probe has the nucleotide sequence of SEQ ID NO:10.
 24. Themethod of claim 19 or 20, wherein each of the firstLactobacillus-specific detection probe, the first G. vaginalis-specificdetection probe, and the first Eggerthella-specific detection probecomprises a label.
 25. The method of claim 22, wherein the secondLactobacillus-specific detection probe comprises a label.
 26. The methodof claim 24 or 25, wherein the label is a chemiluminescent label or afluorescent label.
 27. The method of claim 24 or 25, wherein thedetecting step (3) occurs during the amplifying step (2).
 28. The methodof claim 27, wherein each detection probe comprises a fluorescent labeland a quencher.
 29. The method of claim 28, wherein each detection probeis a molecular torch, a molecular beacon, or a TaqMan detection probe.30. The method of claim 19 or 20, wherein at least one of the firstLactobacillus-specific detection probe, the first G. vaginalis-specificdetection probe, and the first Eggerthella-specific detection probefurther comprises a non-target-hybridizing sequence.
 31. The method ofclaim 30, wherein each of the first Lactobacillus-specific detectionprobe, the first G. vaginalis-specific detection probe, and the firstEggerthella-specific detection probe is a molecular torch or a molecularbeacon.
 32. The method of claim 22, wherein the secondLactobacillus-specific detection probe further comprises anon-target-hybridizing sequence.
 33. The method of claim 32, wherein thesecond Lactobacillus-specific detection probe is a molecular torch or amolecular beacon.
 34. The method of any one of claims 1 to 33, whereinthe amplification reaction at step (2) is an isothermal amplificationreaction.
 35. The method of claim 34, wherein the amplification reactionis a transcription-mediated amplification (TMA) reaction.
 36. The methodof claim 34 or 35, wherein the amplification reaction is a real-timeamplification reaction.
 37. An oligomer combination for determining thepresence or absence of each of Lactobacillus sp., G. vaginalis, andEggerthella sp. in a sample, the oligomer combination comprising: (a)first and second Lactobacillus-specific amplification oligomers foramplifying a target region of a Lactobacillus sp. target nucleic acid,wherein (i) the first Lactobacillus-specific amplification oligomercomprises a first Lactobacillus-specific target-hybridizing sequencesubstantially corresponding to the nucleotide sequence of residues 28-45of SEQ ID NO:7 or residues 28-45 of SEQ ID NO:8 and (ii) the secondLactobacillus-specific amplification oligomer comprises a secondLactobacillus-specific target-hybridizing sequence substantiallycorresponding to the nucleotide sequence of SEQ ID NO:6; (b) first andsecond G. vaginalis-specific amplification oligomers for amplifying atarget region of a G. vaginalis target nucleic acid, wherein (i) thefirst G. vaginalis-specific amplification oligomer comprises a first G.vaginalis-specific target-hybridizing sequence substantiallycorresponding to the nucleotide sequence of residues 28-45 of SEQ IDNO:13 and (ii) the second G. vaginalis-specific amplification oligomercomprises a second G. vaginalis-specific target-hybridizing sequencesubstantially corresponding to the nucleotide sequence of SEQ ID NO:12;and (c) first and second Eggerthella-specific amplification oligomersfor amplifying a target region of an Eggerthella sp. target nucleicacid, wherein (i) the first Eggerthella-specific amplification oligomercomprises a first Eggerthella-specific target-hybridizing sequencesubstantially corresponding to the nucleotide sequence of residues 28-51of SEQ ID NO:17 and (ii) the second Eggerthella-specific amplificationoligomer comprises a second Eggerthella-specific target-hybridizingsequence substantially corresponding to the nucleotide sequence of SEQID NO:16.
 38. The oligomer combination of claim 37, wherein the firstLactobacillus-specific target-hybridizing sequence substantiallycorresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO:7,and wherein the oligomer combination further comprises a thirdLactobacillus-specific amplification oligomer for amplifying theLactobacillus sp. target region, wherein the thirdLactobacillus-specific amplification oligomer comprises a thirdLactobacillus-specific target-hybridizing sequence substantiallycorresponding to the nucleotide sequence of residues 28-45 of SEQ IDNO:8.
 39. The oligomer combination of claim 37, wherein the firstLactobacillus-specific target-hybridizing sequence comprises thenucleotide sequence of residues 28-45 of SEQ ID NO:7 or residues 28-45of SEQ ID NO:8; the second Lactobacillus-specific target-hybridizingsequence comprises the nucleotide sequence of SEQ ID NO:6; the first G.vaginalis-specific target-hybridizing sequence comprises the nucleotidesequence of residues 28-45 of SEQ ID NO:13; the second G.vaginalis-specific target-hybridizing sequence comprises the nucleotidesequence of SEQ ID NO:12; the first Eggerthella-specifictarget-hybridizing sequence comprises the nucleotide sequence ofresidues 28-51 of SEQ ID NO:17; and/or the second Eggerthella-specifictarget-hybridizing sequence comprises the nucleotide sequence of SEQ IDNO:16.
 40. The oligomer combination of claim 37 or 39, wherein the firstLactobacillus-specific target-hybridizing sequence substantiallycorresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO:7,and wherein the oligomer combination further comprises a thirdLactobacillus-specific amplification oligomer for amplifying theLactobacillus sp. target region, wherein the thirdLactobacillus-specific amplification oligomer comprises a thirdLactobacillus-specific target-hybridizing sequence comprising thenucleotide sequence of residues 28-45 of SEQ ID NO:8.
 41. The oligomercombination of claim 37 or 39, wherein the first Lactobacillus-specifictarget-hybridizing sequence consists of the nucleotide sequence ofresidues 28-45 of SEQ ID NO:7 or residues 28-45 of SEQ ID NO:8; thesecond Lactobacillus-specific target-hybridizing sequence consists ofthe nucleotide sequence of SEQ ID NO:6; the first G. vaginalis-specifictarget-hybridizing sequence consists of the nucleotide sequence ofresidues 28-45 of SEQ ID NO:13; the second G. vaginalis-specifictarget-hybridizing sequence consists of the nucleotide sequence of SEQID NO:12; the first Eggerthella-specific target-hybridizing sequenceconsists of the nucleotide sequence of residues 28-51 of SEQ ID NO:17;and/or the second Eggerthella-specific target-hybridizing sequenceconsists of the nucleotide sequence of SEQ ID NO:16.
 42. The oligomercombination of any of claims 37, 39, and 41, wherein the firstLactobacillus-specific target-hybridizing sequence substantiallycorresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO:7,and wherein the oligomer combination further comprises a thirdLactobacillus-specific amplification oligomer for amplifying theLactobacillus sp. target region, wherein the thirdLactobacillus-specific amplification oligomer comprises a thirdLactobacillus-specific target-hybridizing sequence consisting of thenucleotide sequence of residues 28-45 of SEQ ID NO:8.
 43. The oligomercombination of any one of claims 37 to 42, wherein at least one of thefirst Lactobacillus-specific amplification oligomer, the first G.vaginalis-specific amplification oligomer, and the firstEggerthella-specific amplification oligomer is a promoter primer orpromoter provider further comprising a promoter sequence located 5′ tothe respective target hybridizing sequence.
 44. The oligomer combinationof any one of claims 37, 39, 41, and 43, wherein the firstLactobacillus-specific target-hybridizing sequence substantiallycorresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO:7,and wherein oligomer combination further comprises a thirdLactobacillus-specific amplification oligomer for amplifying theLactobacillus sp. target region, wherein the thirdLactobacillus-specific amplification oligomer is a promoter primer orpromoter provider comprising a third Lactobacillus-specifictarget-hybridizing sequence substantially corresponding to thenucleotide sequence of residues 28-45 of SEQ ID NO:8 and furthercomprising a promoter sequence located 5′ to the thirdLactobacillus-specific target hybridizing sequence.
 45. The oligomercombination of any of claims 38, 40, and 42, wherein the thirdLactobacillus-specific amplification oligomer is a promoter primer orpromoter provider further comprising a promoter sequence located 5′ tothe third Lactobacillus-specific target hybridizing sequence.
 46. Theoligomer combination of claim 43 or 45, wherein the promoter sequence isa T7 promoter sequence.
 47. The oligomer combination of claim 46,wherein the promoter sequence has the nucleotide sequence of residues1-27 of SEQ ID NO:7.
 48. The oligomer combination of 43, wherein thefirst Lactobacillus-specific amplification oligomer has the nucleotidesequence of SEQ ID NO:7; the first G. vaginalis-specific amplificationoligomer has the nucleotide sequence of SEQ ID NO:13; and/or the firstEggerthella-specific amplification oligomer has the nucleotide sequenceof SEQ ID NO:17.
 49. The oligomer combination of claim 45, wherein thethird Lactobacillus-specific amplification oligomer has the nucleotidesequence of SEQ ID NO:8.
 50. The oligomer combination of any of claims37 to 49, further comprising at least one capture probe oligomercomprising a target-hybridizing sequence covalently attached to asequence or moiety that binds to an immobilized probe.
 51. The oligomercombination of claim 50, wherein the oligomer combination comprises aLactobacillus-specific capture probe oligomer, a G. vaginalis-specificcapture probe oligomer, and an Eggerthella-specific capture probeoligomer, wherein each of the Lactobacillus-specific, G.vaginalis-specific, and Eggerthella-specific capture probe oligomerscomprises a capture probe target-hybridizing sequence that specificallyhybridizes to a target sequence within the Lactobacillus, G. vaginalis,or Eggerthella target nucleic acid, respectively, and wherein each ofthe Lactobacillus-specific, G. vaginalis-specific, andEggerthella-specific capture probe target-hybridizing sequences iscovalently attached to the sequence or moiety that binds to theimmobilized probe.
 52. The oligomer combination of claim 51, wherein theLactobacillus-specific capture probe target-hybridizing sequencesubstantially corresponds to the nucleotide sequence of residues 1-12 ofSEQ ID NO:5, the G. vaginalis-specific capture probe target-hybridizingsequence substantially corresponds to the nucleotide sequence ofresidues 1-17 of SEQ ID NO:11, and/or the Eggerthella-specific captureprobe target hybridizing sequence substantially corresponds to thenucleotide sequence of residues 1-21 of SEQ ID NO:15.
 53. The oligomercombination of claim 52, wherein the Lactobacillus-specific captureprobe oligomer has the nucleotide sequence of SEQ ID NO:5, the G.vaginalis-specific capture probe oligomer has the nucleotide sequence ofSEQ ID NO:11, and/or the Eggerthella-specific capture probe oligomer hasthe nucleotide sequence of SEQ ID NO:15.
 54. The oligomer combination ofany of claims 37 to 53, further comprising a firstLactobacillus-specific detection probe that specifically hybridizes tothe Lactobacillus sp. target region, a first G. vaginalis-specificdetection probe that specifically hybridizes to the G. vaginalis targetregion, and a first Eggerthella-specific detection probe thatspecifically hybridizes to the Eggerthella sp. target region.
 55. Theoligomer combination of claim 54, wherein the firstLactobacillus-specific detection probe comprises a target-hybridizingsequence substantially corresponding to SEQ ID NO:9, the first G.vaginalis-specific detection probe comprises a target-hybridizingsequence substantially corresponding to residues 1-19 of SEQ ID NO:14,and/or the first Eggerthella-specific detection probe comprises atarget-hybridizing sequence substantially corresponding to SEQ ID NO:18.56. The oligomer combination of claim 55, wherein the firstLactobacillus-specific detection probe has the nucleotide sequence ofSEQ ID NO:9, the first G. vaginalis-specific detection probe has thenucleotide sequence of SEQ ID NO:14, and/or the firstEggerthella-specific detection probe has the nucleotide sequence of SEQID NO:18.
 57. The oligomer combination of claim 55 or 56, furthercomprising a second Lactobacillus-specific detection probe thatspecifically hybridizes to the Lactobacillus sp. target region, whereinthe second Lactobacillus-specific detection probe comprises atarget-hybridizing sequence substantially corresponding to residues 6-21of SEQ ID NO:10.
 58. The oligomer combination of claim 57, wherein thesecond Lactobacillus-specific detection probe has the nucleotidesequence of SEQ ID NO:10.
 59. The oligomer combination of claim 54 or55, wherein each of the first Lactobacillus-specific detection probe,the first G. vaginalis-specific detection probe, and the firstEggerthella-specific detection probe comprises a label.
 60. The oligomercombination of claim 57, wherein the second Lactobacillus-specificdetection probe comprises a label.
 61. The oligomer combination of claim59 or 60, wherein the label is a chemiluminescent label or a fluorescentlabel.
 62. The oligomer combination of claim 59 or 60, wherein eachdetection probe comprises a fluorescent label and a quencher.
 63. Theoligomer combination of claim 62, wherein each detection probe is amolecular torch, a molecular beacon, or a TaqMan detection probe. 64.The oligomer combination of claim 54 or 55, wherein at least one of thefirst Lactobacillus-specific detection probe, the first G.vaginalis-specific detection probe, and the first Eggerthella-specificdetection probe further comprises a non-target-hybridizing sequence. 65.The oligomer combination of claim 64, wherein each of the firstLactobacillus-specific detection probe, the first G. vaginalis-specificdetection probe, and the first Eggerthella-specific detection probe is amolecular torch or a molecular beacon.
 66. The oligomer combination ofclaim 57, wherein the second Lactobacillus-specific detection probefurther comprises a non-target-hybridizing sequence.
 67. The oligomercombination of claim 66, wherein the second Lactobacillus-specificdetection probe is a molecular torch or a molecular beacon.
 68. A methodfor determining the presence or absence of Bacterial Vaginosis (BV) in asubject, the method comprising: (a) providing a sample from a subjectsuspected of having BV; (b) performing an assay for the detection ofLactobacillus sp., G. vaginalis, and Eggerthella sp. in the sample; (c)for each of Lactobacillus sp., G. vaginalis, and Eggerthella sp.,assigning a status of either positive or negative based on the detectionassay; and (d) determining the presence or absence of BV in the subjectbased on a combination of the assigned Lactobacillus sp. status, G.vaginalis status, and Eggerthella sp. status from step (c), wherein (i)a negative status for both G. vaginalis and Eggerthella sp. indicatesthe absence of BV in the subject, (ii) a positive status for both G.vaginalis and Eggerthella sp. indicates the presence of BV in thesubject, (iii) if the status of Lactobacillus sp. is positive, then anegative status for at least one of G. vaginalis and Eggerthellaindicates the absence of BV in the subject, and (iv) if the status ofLactobacillus sp. is negative, then a positive status for at least oneof G. vaginalis and Eggerthella indicates the presence of BV in thesubject.
 69. The method of claim 68, wherein the assay for detection ofLactobacillus sp., G. vaginalis, and Eggerthella sp. is anucleic-acid-based detection assay.
 70. The method of claim 69, whereinthe nucleic-acid-based detection assay targets the 16S rRNA ofLactobacillus sp., G. vaginalis, and Eggerthella sp.
 71. The method ofclaim 70, wherein the nucleic-acid-based detection assay targets (i) aLactobacillus sp. 16S rRNA region corresponding to a region of SEQ IDNO:1 from about nucleotide position 91 to about nucleotide position 265;(ii) a G. vaginalis 16S rRNA region corresponding to a region of SEQ IDNO:3 from about nucleotide position 964 to about nucleotide position1036; and/or (iii) an Eggerthella sp. 16S rRNA region corresponding to aregion of SEQ ID NO:4 from about nucleotide position 165 to aboutnucleotide position
 259. 72. The method of any of claims 70 to 71,wherein the nucleic-acid-based detection assay is an amplification-basedassay.
 73. The method of claim 72, wherein the amplification-based assaycomprises an isothermal amplification reaction.
 74. The method of claim73, wherein the amplification reaction is a transcription-mediatedamplification (TMA) reaction.
 75. The method of claim 73 or 74, whereinthe amplification reaction is a real-time amplification reaction. 76.The method of claim 70, wherein the nucleic-acid-based detection assayis an amplification-based assay comprising (1) contacting the samplewith first and second Lactobacillus-specific amplification oligomers foramplifying a target region of a Lactobacillus sp. target nucleic acid,wherein (i) the first Lactobacillus-specific amplification oligomercomprises a first Lactobacillus-specific target-hybridizing sequencesubstantially corresponding to the nucleotide sequence of residues 28-45of SEQ ID NO:7 or residues 28-45 of SEQ ID NO:8 and (ii) the secondLactobacillus-specific amplification oligomer comprises a secondLactobacillus-specific target-hybridizing sequence substantiallycorresponding to the nucleotide sequence of SEQ ID NO:6; first andsecond G. vaginalis-specific amplification oligomers for amplifying atarget region of a G. vaginalis target nucleic acid, wherein (i) thefirst G. vaginalis-specific amplification oligomer comprises a first G.vaginalis-specific target-hybridizing sequence substantiallycorresponding to the nucleotide sequence of residues 28-45 of SEQ IDNO:13 and (ii) the second G. vaginalis-specific amplification oligomercomprises a second G. vaginalis-specific target-hybridizing sequencesubstantially corresponding to the nucleotide sequence of SEQ ID NO:12;and first and second Eggerthella-specific amplification oligomers foramplifying a target region of an Eggerthella sp. target nucleic acid,wherein (i) the first Eggerthella-specific amplification oligomercomprises a first Eggerthella-specific target-hybridizing sequencesubstantially corresponding to the nucleotide sequence of residues 28-51of SEQ ID NO:17 and (ii) the second Eggerthella-specific amplificationoligomer comprises a second Eggerthella-specific target-hybridizingsequence substantially corresponding to the nucleotide sequence of SEQID NO:16; (2) performing an in vitro nucleic acid amplificationreaction, wherein any Lactobacillus sp., G. vaginalis, and Eggerthellasp. target nucleic acid, if present in the sample, is used as a templatefor generating one or more amplification products corresponding to theLactobacillus sp., G. vaginalis, and Eggerthella sp. target regions; and(3) detecting the presence or absence of the one or more amplificationproducts.
 77. The method of claim 76, wherein the firstLactobacillus-specific target-hybridizing sequence substantiallycorresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO:7,and wherein the sample is further contacted with a thirdLactobacillus-specific amplification oligomer for amplifying theLactobacillus sp. target region, wherein the thirdLactobacillus-specific amplification oligomer comprises a thirdLactobacillus-specific target-hybridizing sequence substantiallycorresponding to the nucleotide sequence of residues 28-45 of SEQ IDNO:8.
 78. The method of claim 76, wherein the firstLactobacillus-specific target-hybridizing sequence comprises thenucleotide sequence of residues 28-45 of SEQ ID NO:7 or residues 28-45of SEQ ID NO:8; the second Lactobacillus-specific target-hybridizingsequence comprises the nucleotide sequence of SEQ ID NO:6; the first G.vaginalis-specific target-hybridizing sequence comprises the nucleotidesequence of residues 28-45 of SEQ ID NO:13; the second G.vaginalis-specific target-hybridizing sequence comprises the nucleotidesequence of SEQ ID NO:12; the first Eggerthella-specifictarget-hybridizing sequence comprises the nucleotide sequence ofresidues 28-51 of SEQ ID NO:17; and/or the second Eggerthella-specifictarget-hybridizing sequence comprises the nucleotide sequence of SEQ IDNO:16.
 79. The method of claim 76 or 78, wherein the firstLactobacillus-specific target-hybridizing sequence substantiallycorresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO:7,and wherein the sample is further contacted with a thirdLactobacillus-specific amplification oligomer for amplifying theLactobacillus sp. target region, wherein the thirdLactobacillus-specific amplification oligomer comprises a thirdLactobacillus-specific target-hybridizing sequence comprising thenucleotide sequence of residues 28-45 of SEQ ID NO:8.
 80. The method ofclaim 76 or 78, wherein the first Lactobacillus-specifictarget-hybridizing sequence consists of the nucleotide sequence ofresidues 28-45 of SEQ ID NO:7 or residues 28-45 of SEQ ID NO:8; thesecond Lactobacillus-specific target-hybridizing sequence consists ofthe nucleotide sequence of SEQ ID NO:6; the first G. vaginalis-specifictarget-hybridizing sequence consists of the nucleotide sequence ofresidues 28-45 of SEQ ID NO:13; the second G. vaginalis-specifictarget-hybridizing sequence consists of the nucleotide sequence of SEQID NO:12; the first Eggerthella-specific target-hybridizing sequenceconsists of the nucleotide sequence of residues 28-51 of SEQ ID NO:17;and/or the second Eggerthella-specific target-hybridizing sequenceconsists of the nucleotide sequence of SEQ ID NO:16.
 81. The method ofany of claims 76, 78, and 80, wherein the first Lactobacillus-specifictarget-hybridizing sequence substantially corresponds to the nucleotidesequence of residues 28-45 of SEQ ID NO:7, and wherein the sample isfurther contacted with a third Lactobacillus-specific amplificationoligomer for amplifying the Lactobacillus sp. target region, wherein thethird Lactobacillus-specific amplification oligomer comprises a thirdLactobacillus-specific target-hybridizing sequence consisting of thenucleotide sequence of residues 28-45 of SEQ ID NO:8.
 82. The method ofany one of claims 76 to 81, wherein at least one of the firstLactobacillus-specific amplification oligomer, the first G.vaginalis-specific amplification oligomer, and the firstEggerthella-specific amplification oligomer is a promoter primer orpromoter provider further comprising a promoter sequence located 5′ tothe respective target hybridizing sequence.
 83. The method of any one ofclaims 76, 78, 80, and 82, wherein the first Lactobacillus-specifictarget-hybridizing sequence substantially corresponds to the nucleotidesequence of residues 28-45 of SEQ ID NO:7, and wherein the sample isfurther contacted with a third Lactobacillus-specific amplificationoligomer for amplifying the Lactobacillus sp. target region, wherein thethird Lactobacillus-specific amplification oligomer is a promoter primeror promoter provider comprising a third Lactobacillus-specifictarget-hybridizing sequence substantially corresponding to thenucleotide sequence of residues 28-45 of SEQ ID NO:8 and furthercomprising a promoter sequence located 5′ to the thirdLactobacillus-specific target hybridizing sequence.
 84. The method ofany of claims 77, 79, and 81, wherein the third Lactobacillus-specificamplification oligomer is a promoter primer or promoter provider furthercomprising a promoter sequence located 5′ to the thirdLactobacillus-specific target hybridizing sequence.
 85. The method ofclaim 82 or 84, wherein the promoter sequence is a T7 promoter sequence.86. The method of claim 85, wherein the promoter sequence has thenucleotide sequence of residues 1-27 of SEQ ID NO:7.
 87. The method ofclaim 82, wherein the first Lactobacillus-specific amplificationoligomer has the nucleotide sequence of SEQ ID NO:7; the first G.vaginalis-specific amplification oligomer has the nucleotide sequence ofSEQ ID NO:13; and/or the first Eggerthella-specific amplificationoligomer has the nucleotide sequence of SEQ ID NO:17.
 88. The method ofclaim 84, wherein the third Lactobacillus-specific amplificationoligomer has the nucleotide sequence of SEQ ID NO:8.
 89. The method ofany one of claims 76 to 88, further comprising purifying theLactobacillus, G. vaginalis, and Eggerthella target nucleic acids, ifpresent, from other components in the sample before step (2).
 90. Themethod of claim 89, wherein the purifying step comprises contacting thesample with at least one capture probe oligomer comprising atarget-hybridizing sequence covalently attached to a sequence or moietythat binds to an immobilized probe.
 91. The method of claim 90, whereinthe sample is contacted with a Lactobacillus-specific capture probeoligomer, a G. vaginalis-specific capture probe oligomer, and anEggerthella-specific capture probe oligomer, wherein each of theLactobacillus-specific, G. vaginalis-specific, and Eggerthella-specificcapture probe oligomers comprises a capture probe target-hybridizingsequence that specifically hybridizes to a target sequence within theLactobacillus, G. vaginalis, or Eggerthella target nucleic acid,respectively, and wherein each of the Lactobacillus-specific, G.vaginalis-specific, and Eggerthella-specific capture probetarget-hybridizing sequences is covalently attached to the sequence ormoiety that binds to the immobilized probe.
 92. The method of claim 91,wherein the Lactobacillus-specific capture probe target-hybridizingsequence substantially corresponds to the nucleotide sequence ofresidues 1-12 of SEQ ID NO:5, the G. vaginalis-specific capture probetarget-hybridizing sequence substantially corresponds to the nucleotidesequence of residues 1-17 of SEQ ID NO:11, and/or theEggerthella-specific capture probe target hybridizing sequencesubstantially corresponds to the nucleotide sequence of residues 1-21 ofSEQ ID NO:15.
 93. The method of claim 92, wherein theLactobacillus-specific capture probe oligomer has the nucleotidesequence of SEQ ID NO:5, the G. vaginalis-specific capture probeoligomer has the nucleotide sequence of SEQ ID NO:11, and/or theEggerthella-specific capture probe oligomer has the nucleotide sequenceof SEQ ID NO:15.
 94. The method of any of claims 76 to 93, wherein thedetecting step (3) comprises contacting the one or more amplificationproducts with a first Lactobacillus-specific detection probe thatspecifically hybridizes to the Lactobacillus sp. target region, a firstG. vaginalis-specific detection probe that specifically hybridizes tothe G. vaginalis target region, and a first Eggerthella-specificdetection probe that specifically hybridizes to the Eggerthella sp.target region, and detecting the presence or absence of anytarget-hybridized Lactobacillus-specific, G. vaginalis-specific, and/orEggerthella-specific detection probe.
 95. The method of claim 94,wherein the first Lactobacillus-specific detection probe comprises atarget-hybridizing sequence substantially corresponding to SEQ ID NO:9,the first G. vaginalis-specific detection probe comprises atarget-hybridizing sequence substantially corresponding to residues 1-19of SEQ ID NO:14, and/or the first Eggerthella-specific detection probecomprises a target-hybridizing sequence substantially corresponding toSEQ ID NO:18.
 96. The method of claim 95, wherein the firstLactobacillus-specific detection probe has the nucleotide sequence ofSEQ ID NO:9, the first G. vaginalis-specific detection probe has thenucleotide sequence of SEQ ID NO:14, and/or the firstEggerthella-specific detection probe has the nucleotide sequence of SEQID NO:18.
 97. The method of claim 95 or 96, wherein the detecting step(3) further comprises contacting the one or more amplification productswith a second Lactobacillus-specific detection probe that specificallyhybridizes to the Lactobacillus sp. target region, wherein the secondLactobacillus-specific detection probe comprises a target-hybridizingsequence substantially corresponding to residues 6-21 of SEQ ID NO:10.98. The method of claim 97, wherein the second Lactobacillus-specificdetection probe has the nucleotide sequence of SEQ ID NO:10.
 99. Themethod of claim 94 or 95, wherein each of the firstLactobacillus-specific detection probe, the first G. vaginalis-specificdetection probe, and the first Eggerthella-specific detection probecomprises a label.
 100. The method of claim 97, wherein the secondLactobacillus-specific detection probe comprises a label.
 101. Themethod of claim 99 or 100, wherein the label is a chemiluminescent labelor a fluorescent label.
 102. The method of claim 99 or 100, wherein thedetecting step (3) occurs during the amplifying step (2).
 103. Themethod of claim 102, wherein each detection probe comprises afluorescent label and a quencher.
 104. The method of claim 103, whereineach detection probe is a molecular torch, a molecular beacon, or aTaqMan detection probe.
 105. The method of claim 94 or 95, wherein atleast one of the first Lactobacillus-specific detection probe, the firstG. vaginalis-specific detection probe, and the firstEggerthella-specific detection probe further comprises anon-target-hybridizing sequence.
 106. The method of claim 105, whereineach of the first Lactobacillus-specific detection probe, the first G.vaginalis-specific detection probe, and the first Eggerthella-specificdetection probe is a molecular torch or a molecular beacon.
 107. Themethod of claim 97, wherein the second Lactobacillus-specific detectionprobe further comprises a non-target-hybridizing sequence.
 108. Themethod of claim 107, wherein the second Lactobacillus-specific detectionprobe is a molecular torch or a molecular beacon.
 109. The method of anyone of claims 76 to 108, wherein the amplification reaction at step (2)is an isothermal amplification reaction.
 110. The method of claim 109,wherein the amplification reaction is a transcription-mediatedamplification (TMA) reaction.
 111. The method of claim 109 or 110,wherein the amplification reaction is a real-time amplificationreaction.
 112. The method of any of claims 68 to 111, wherein the methodcomprises the detection of no more than ten bacterial genera associatedwith BV.
 113. The method of any of claims 68 to 111, wherein the methodcomprises the detection of no more than five bacterial genera associatedwith BV.
 114. The method of any of claims 68 to 111, wherein the methoddoes not include detection of bacterial genera associated with BV otherthan Lactobacillus, Gardnerella, and Eggerthella.
 115. The method of anyof claims 68 to 114, wherein if the presence of BV is indicated in thesubject, then the method further comprises administering a treatmentregime for BV to the subject.
 116. The method of any of claims 68 to114, wherein the method is a method for monitoring BV in the subject andthe subject is undergoing a treatment regime for BV prior to step (a).117. The method of claim 116, wherein if the presence of BV is indicatedin the subject, then the method further comprises either (i)administering the treatment regime for BV to the subject or (ii)administering a different treatment regime for BV to the subject.
 118. Amethod for determining the presence or absence of Lactobacillus sp. in asample, the method comprising: (1) contacting a sample, said samplesuspected of containing Lactobacillus sp., with first and secondLactobacillus-specific amplification oligomers for amplifying a targetregion of a Lactobacillus sp. target nucleic acid, wherein the first andsecond Lactobacillus-specific amplification oligomers respectivelycomprise first and second Lactobacillus-specific target-hybridizingsequences, and wherein the first and second amplification oligomerstarget a Lactobacillus sp. 16S rRNA region corresponding to a region ofSEQ ID NO:1 from about nucleotide position 91 to about nucleotideposition 265; (2) performing an in vitro nucleic acid amplificationreaction, wherein any Lactobacillus sp. target nucleic acid, if presentin the sample, is used as a template for generating one or moreamplification products corresponding to the Lactobacillus sp. targetregion; and (3) detecting the presence or absence of the one or moreamplification products, thereby determining the presence or absence ofLactobacillus sp. in the sample.
 119. The method of claim 118, wherein(i) the first Lactobacillus-specific target-hybridizing sequencesubstantially corresponds to the nucleotide sequence of residues 28-45of SEQ ID NO:7 or residues 28-45 of SEQ ID NO:8 and (ii) the secondLactobacillus-specific target-hybridizing sequence substantiallycorresponds to the nucleotide sequence of SEQ ID NO:6.
 120. The methodof claim 118 or 119, wherein the first Lactobacillus-specifictarget-hybridizing sequence substantially corresponds to the nucleotidesequence of residues 28-45 of SEQ ID NO:7, and wherein the sample isfurther contacted with a third Lactobacillus-specific amplificationoligomer for amplifying the Lactobacillus sp. target region, wherein thethird Lactobacillus-specific amplification oligomer comprises a thirdLactobacillus-specific target-hybridizing sequence substantiallycorresponding to the nucleotide sequence of residues 28-45 of SEQ IDNO:8.
 121. The method of claim 118 or 119, wherein the firstLactobacillus-specific target-hybridizing sequence comprises thenucleotide sequence of residues 28-45 of SEQ ID NO:7 or residues 28-45of SEQ ID NO:8; and/or the second Lactobacillus-specifictarget-hybridizing sequence comprises the nucleotide sequence of SEQ IDNO:6.
 122. The method of claim 118, 119, or 121, wherein the firstLactobacillus-specific target-hybridizing sequence substantiallycorresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO:7,and wherein the sample is further contacted with a thirdLactobacillus-specific amplification oligomer for amplifying theLactobacillus sp. target region, wherein the thirdLactobacillus-specific amplification oligomer comprises a thirdLactobacillus-specific target-hybridizing sequence comprising thenucleotide sequence of residues 28-45 of SEQ ID NO:8.
 123. The method ofclaim 118, 119, or 121, wherein the first Lactobacillus-specifictarget-hybridizing sequence consists of the nucleotide sequence ofresidues 28-45 of SEQ ID NO:7 or residues 28-45 of SEQ ID NO:8; and/orthe second Lactobacillus-specific target-hybridizing sequence consistsof the nucleotide sequence of SEQ ID NO:6.
 124. The method of any ofclaim 118, 119, 121, or 123, wherein the first Lactobacillus-specifictarget-hybridizing sequence substantially corresponds to the nucleotidesequence of residues 28-45 of SEQ ID NO:7, and wherein the sample isfurther contacted with a third Lactobacillus-specific amplificationoligomer for amplifying the Lactobacillus sp. target region, wherein thethird Lactobacillus-specific amplification oligomer comprises a thirdLactobacillus-specific target-hybridizing sequence consisting of thenucleotide sequence of residues 28-45 of SEQ ID NO:8.
 125. The method ofany one of claims 118 to 124, wherein the first Lactobacillus-specificamplification oligomer, the first G. vaginalis-specific amplificationoligomer is a promoter primer or promoter provider further comprising apromoter sequence located 5′ to the first Lactabacillus-specific targethybridizing sequence.
 126. The method of any one of claims 118, 119,121, 123, and 125, wherein the first Lactobacillus-specifictarget-hybridizing sequence substantially corresponds to the nucleotidesequence of residues 28-45 of SEQ ID NO:7, and wherein the sample isfurther contacted with a third Lactobacillus-specific amplificationoligomer for amplifying the Lactobacillus sp. target region, wherein thethird Lactobacillus-specific amplification oligomer is a promoter primeror promoter provider comprising a third Lactobacillus-specifictarget-hybridizing sequence substantially corresponding to thenucleotide sequence of residues 28-45 of SEQ ID NO:8 and furthercomprising a promoter sequence located 5′ to the thirdLactobacillus-specific target hybridizing sequence.
 127. The method ofany of claims 120, 122, and 124, wherein the thirdLactobacillus-specific amplification oligomer is a promoter primer orpromoter provider further comprising a promoter sequence located 5′ tothe third Lactobacillus-specific target hybridizing sequence.
 128. Themethod of claim 125 or 127, wherein the promoter sequence is a T7promoter sequence.
 129. The method of claim 128, wherein the promotersequence has the nucleotide sequence of residues 1-27 of SEQ ID NO:7.130. The method of claim 125, wherein the first Lactobacillus-specificamplification oligomer has the nucleotide sequence of SEQ ID NO:7. 131.The method of claim 127, wherein the third Lactobacillus-specificamplification oligomer has the nucleotide sequence of SEQ ID NO:8. 132.The method of any one of claims 118 to 131, further comprising purifyingthe Lactobacillus target nucleic acid, if present, from other componentsin the sample before step (2).
 133. The method of claim 132, wherein thepurifying step comprises contacting the sample with at least one captureprobe oligomer comprising a target-hybridizing sequence covalentlyattached to a sequence or moiety that binds to an immobilized probe.134. The method of claim 133, wherein the sample is contacted with aLactobacillus-specific capture probe oligomer comprising a capture probetarget-hybridizing sequence that specifically hybridizes to a targetsequence within the Lactobacillus target nucleic acid, and wherein theLactobacillus-specific capture probe target-hybridizing sequence iscovalently attached to the sequence or moiety that binds to theimmobilized probe.
 135. The method of claim 134, wherein theLactobacillus-specific capture probe target-hybridizing sequencesubstantially corresponds to the nucleotide sequence of residues 1-12 ofSEQ ID NO:5.
 136. The method of claim 135, wherein theLactobacillus-specific capture probe oligomer has the nucleotidesequence of SEQ ID NO:5.
 137. The method of any of claims 118 to 136,wherein the detecting step (3) comprises contacting the one or moreamplification products with a first Lactobacillus-specific detectionprobe that specifically hybridizes to the Lactobacillus sp. targetregion, and detecting the presence or absence of any target-hybridizedLactobacillus-specific detection probe.
 138. The method of claim 137,wherein the first Lactobacillus-specific detection probe comprises atarget-hybridizing sequence substantially corresponding to SEQ ID NO:9.139. The method of claim 138, wherein the first Lactobacillus-specificdetection probe has the nucleotide sequence of SEQ ID NO:9.
 140. Themethod of claim 138 or 139, wherein the detecting step (3) furthercomprises contacting the one or more amplification products with asecond Lactobacillus-specific detection probe that specificallyhybridizes to the Lactobacillus sp. target region, wherein the secondLactobacillus-specific detection probe comprises a target-hybridizingsequence substantially corresponding to residues 6-21 of SEQ ID NO:10.141. The method of claim 140, wherein the second Lactobacillus-specificdetection probe has the nucleotide sequence of SEQ ID NO:10.
 142. Themethod of claim 137 or 138, wherein the first Lactobacillus-specificdetection probe comprises a label.
 143. The method of claim 140, whereinthe second Lactobacillus-specific detection probe comprises a label.144. The method of claim 142 or 143, wherein the label is achemiluminescent label or a fluorescent label.
 145. The method of claim142 or 143, wherein the detecting step (3) occurs during the amplifyingstep (2).
 146. The method of claim 145, wherein each detection probecomprises a fluorescent label and a quencher.
 147. The method of claim146, wherein each detection probe is a molecular torch, a molecularbeacon, or a TaqMan detection probe.
 148. The method of claim 137,wherein the first Lactobacillus-specific detection probe furthercomprises a non-target-hybridizing sequence.
 149. The method of claim137 or 138, wherein the first Lactobacillus-specific detection probe isa molecular torch or a molecular beacon.
 150. The method of claim 140,wherein the second Lactobacillus-specific detection probe furthercomprises a non-target-hybridizing sequence.
 151. The method of claim140 or 150, wherein the second Lactobacillus-specific detection probe isa molecular torch or a molecular beacon.
 152. A method for determiningthe presence or absence of G. vaginalis in a sample, the methodcomprising: (1) contacting a sample, said sample suspected of containingG. vaginalis, with first and second G. vaginalis-specific amplificationoligomers for amplifying a target region of a G. vaginalis targetnucleic acid, wherein the first and second G. vaginalis-specificamplification oligomers respectively comprise first and second G.vaginalis-specific target hybridizing sequences, and wherein the firstand second amplification oligomers target a G. vaginalis 16S rRNA regioncorresponding to a region of SEQ ID NO:3 from about nucleotide position964 to about nucleotide position 1036; (2) performing an in vitronucleic acid amplification reaction, wherein any G. vaginalis targetnucleic acid, if present in the sample, is used as a template forgenerating one or more amplification products corresponding to the G.vaginalis target region; and (3) detecting the presence or absence ofthe one or more amplification products, thereby determining the presenceor absence of G. vaginalis in the sample.
 153. The method of claim 152,wherein (i) the first G. vaginalis-specific target-hybridizing sequencesubstantially corresponds to the nucleotide sequence of residues 28-45of SEQ ID NO:13 and (ii) the second G. vaginalis-specifictarget-hybridizing sequence substantially corresponds to the nucleotidesequence of SEQ ID NO:12.
 154. The method of claim 152 or 153, whereinthe first G. vaginalis-specific target-hybridizing sequence comprisesthe nucleotide sequence of residues 28-45 of SEQ ID NO:13; and/or thesecond G. vaginalis-specific target-hybridizing sequence comprises thenucleotide sequence of SEQ ID NO:12;
 155. The method of any one ofclaims 152 to 154, wherein the first G. vaginalis-specifictarget-hybridizing sequence consists of the nucleotide sequence ofresidues 28-45 of SEQ ID NO:13; and/or the second G. vaginalis-specifictarget-hybridizing sequence consists of the nucleotide sequence of SEQID NO:12.
 155. The method of any one of claims 152 to 155, wherein thefirst G. vaginalis-specific amplification oligomer is a promoter primeror promoter provider further comprising a promoter sequence located 5′to the first G. vaginalis-specific target hybridizing sequence.
 156. Themethod of claim 155, wherein the promoter sequence is a T7 promotersequence.
 157. The method of claim 156, wherein the promoter sequencehas the nucleotide sequence of residues 1-27 of SEQ ID NO:7.
 158. Themethod of claim 155, wherein the first G. vaginalis-specificamplification oligomer has the nucleotide sequence of SEQ ID NO:13. 159.The method of any one of claims 152 to 158, further comprising purifyingthe G. vaginalis target nucleic acid, if present, from other componentsin the sample before step (2).
 160. The method of claim 159, wherein thepurifying step comprises contacting the sample with at least one captureprobe oligomer comprising a target-hybridizing sequence covalentlyattached to a sequence or moiety that binds to an immobilized probe.161. The method of claim 160, wherein the sample is contacted with a G.vaginalis-specific capture probe oligomer comprising a capture probetarget-hybridizing sequence that specifically hybridizes to a targetsequence within the G. vaginalis target nucleic acid, wherein the G.vaginalis-specific capture probe target-hybridizing sequence iscovalently attached to the sequence or moiety that binds to theimmobilized probe.
 162. The method of claim 161, wherein the G.vaginalis-specific capture probe target-hybridizing sequencesubstantially corresponds to the nucleotide sequence of residues 1-17 ofSEQ ID NO:11.
 163. The method of claim 162, wherein the G.vaginalis-specific capture probe oligomer has the nucleotide sequence ofSEQ ID NO:11.
 164. The method of any of claims 152 to 163, wherein thedetecting step (3) comprises contacting the one or more amplificationproducts with a first G. vaginalis-specific detection probe thatspecifically hybridizes to the G. vaginalis target region, and detectingthe presence or absence of any target-hybridized G. vaginalis-specificdetection probe.
 165. The method of claim 164, wherein the first G.vaginalis-specific detection probe comprises a target-hybridizingsequence substantially corresponding to residues 1-19 of SEQ ID NO:14.166. The method of claim 165, wherein the first G. vaginalis-specificdetection probe has the nucleotide sequence of SEQ ID NO:14.
 167. Themethod of claim 164 or 165, wherein the first G. vaginalis-specificdetection probe detection probe comprises a label.
 168. The method ofclaim 167, wherein the label is a chemiluminescent label or afluorescent label.
 169. The method of claim 167, wherein the detectingstep (3) occurs during the amplifying step (2).
 170. The method of claim169, wherein the detection probe comprises a fluorescent label and aquencher.
 171. The method of claim 170, wherein the detection probe is amolecular torch, a molecular beacon, or a TaqMan detection probe. 172.The method of claim 164 or 165, wherein the first G. vaginalis-specificdetection probe further comprises a non-target-hybridizing sequence.173. The method of claim 164 or 172, wherein the first G.vaginalis-specific detection probe is a molecular torch or a molecularbeacon.
 174. A method for determining the presence or absence ofEggerthella sp. in a sample, the method comprising: (1) contacting asample, said sample suspected of containing Eggerthella sp., with firstand second Eggerthella-specific amplification oligomers for amplifying atarget region of a Eggerthella sp. target nucleic acid, wherein thefirst and second Eggerthella-specific amplification oligomersrespectively comprise first and second Eggerthella-specifictarget-hybridizing sequences, and wherein the first and secondamplification oligomers target a Eggerthella sp. 16S rRNA regioncorresponding to a region of SEQ ID NO:4 from about nucleotide position165 to about nucleotide position 259; (2) performing an in vitro nucleicacid amplification reaction, wherein any Eggerthella sp. target nucleicacid, if present in the sample, is used as a template for generating oneor more amplification products corresponding to the Eggerthella sp.target region; and (3) detecting the presence or absence of the one ormore amplification products, thereby determining the presence or absenceof Eggerthella sp. in the sample.
 175. The method of claim 174, wherein(i) the first Eggerthella-specific target-hybridizing sequencesubstantially corresponds to the nucleotide sequence of residues 28-51of SEQ ID NO:17 and (ii) the second Eggerthella-specifictarget-hybridizing sequence substantially corresponds to the nucleotidesequence of SEQ ID NO:16.
 176. The method of claim 174 or 175, whereinthe first Eggerthella-specific target-hybridizing sequence comprises thenucleotide sequence of residues 28-51 of SEQ ID NO:17; and/or the secondEggerthella-specific target-hybridizing sequence comprises thenucleotide sequence of SEQ ID NO:16.
 177. The method of any one ofclaims 174 to 176, wherein the first Eggerthella-specifictarget-hybridizing sequence consists of the nucleotide sequence ofresidues 28-51 of SEQ ID NO:17; and/or the second Eggerthella-specifictarget-hybridizing sequence consists of the nucleotide sequence of SEQID NO:16.
 178. The method of any one of claims 174 to 177, wherein thefirst Eggerthella-specific amplification oligomer is a promoter primeror promoter provider further comprising a promoter sequence located 5′to the respective target hybridizing sequence.
 179. The method of claim178, wherein the promoter sequence is a T7 promoter sequence.
 180. Themethod of claim 179, wherein the promoter sequence has the nucleotidesequence of residues 1-27 of SEQ ID NO:7.
 181. The method of claim 178,wherein the first Eggerthella-specific amplification oligomer has thenucleotide sequence of SEQ ID NO:17.
 182. The method of any one ofclaims 174 to 181 further comprising purifying the Eggerthella targetnucleic acid, if present, from other components in the sample beforestep (2).
 183. The method of claim 182, wherein the purifying stepcomprises contacting the sample with at least one capture probe oligomercomprising a target-hybridizing sequence covalently attached to asequence or moiety that binds to an immobilized probe.
 184. The methodof claim 183, wherein the sample is contacted with anEggerthella-specific capture probe oligomer comprising a capture probetarget-hybridizing sequence that specifically hybridizes to a targetsequence within the Eggerthella target nucleic acid, wherein theEggerthella-specific capture probe target-hybridizing sequence iscovalently attached to the sequence or moiety that binds to theimmobilized probe.
 185. The method of claim 184, wherein theEggerthella-specific capture probe target hybridizing sequencesubstantially corresponds to the nucleotide sequence of residues 1-21 ofSEQ ID NO:15.
 186. The method of claim 185, wherein theEggerthella-specific capture probe oligomer has the nucleotide sequenceof SEQ ID NO:15.
 187. The method of any of claims 174 to 186, whereinthe detecting step (3) comprises contacting the one or moreamplification products with a first Eggerthella-specific detection probethat specifically hybridizes to the Eggerthella sp. target region, anddetecting the presence or absence of any target-hybridizedEggerthella-specific detection probe.
 188. The method of claim 187,wherein the first Eggerthella-specific detection probe comprises atarget-hybridizing sequence substantially corresponding to SEQ ID NO:18.189. The method of claim 188, wherein the first Eggerthella-specificdetection probe has the nucleotide sequence of SEQ ID NO:18.
 190. Themethod of claim 187 or 188, wherein the first Eggerthella-specificdetection probe comprises a label.
 191. The method of claim 190, whereinthe label is a chemiluminescent label or a fluorescent label.
 192. Themethod of claim 190, wherein the detecting step (3) occurs during theamplifying step (2).
 193. The method of claim 192, wherein the detectionprobe comprises a fluorescent label and a quencher.
 194. The method ofclaim 193, wherein the detection probe is a molecular torch, a molecularbeacon, or a TaqMan detection probe.
 195. The method of claim 187,wherein the first Eggerthella-specific detection probe further comprisesa non-target-hybridizing sequence.
 196. The method of claim 187 or 195,wherein the first Eggerthella-specific detection probe is a moleculartorch or a molecular beacon.
 197. The method of any one of claims 118 to196, wherein the amplification reaction at step (2) is an isothermalamplification reaction.
 198. The method of claim 197, wherein theamplification reaction is a transcription-mediated amplification (TMA)reaction.
 199. The method of claim 197 or 198, wherein the amplificationreaction is a real-time amplification reaction.
 200. A multiplex methodfor determining the presence or absence of each of Lactobacillus sp. andG. vaginalis in a sample, the method comprising: (1) contacting asample, said sample suspected of containing at least one ofLactobacillus sp. and G. vaginalis, with (a) first and secondLactobacillus-specific amplification oligomers for amplifying a targetregion of a Lactobacillus sp. target nucleic acid, wherein (i) the firstLactobacillus-specific amplification oligomer comprises a firstLactobacillus-specific target-hybridizing sequence substantiallycorresponding to the nucleotide sequence of residues 28-45 of SEQ IDNO:7 or residues 28-45 of SEQ ID NO:8 and (ii) the secondLactobacillus-specific amplification oligomer comprises a secondLactobacillus-specific target-hybridizing sequence substantiallycorresponding to the nucleotide sequence of SEQ ID NO:6; and (b) firstand second G. vaginalis-specific amplification oligomers for amplifyinga target region of a G. vaginalis target nucleic acid, wherein (i) thefirst G. vaginalis-specific amplification oligomer comprises a first G.vaginalis-specific target-hybridizing sequence substantiallycorresponding to the nucleotide sequence of residues 28-45 of SEQ IDNO:13 and (ii) the second G. vaginalis-specific amplification oligomercomprises a second G. vaginalis-specific target-hybridizing sequencesubstantially corresponding to the nucleotide sequence of SEQ ID NO:12;(2) performing an in vitro nucleic acid amplification reaction, whereinany Lactobacillus sp. and G. vaginalis target nucleic acid, if presentin the sample, is used as a template for generating one or moreamplification products corresponding to the Lactobacillus sp. and G.vaginalis target regions; and (3) detecting the presence or absence ofthe one or more amplification products, thereby determining the presenceor absence of Lactobacillus sp. and G. vaginalis in the sample.
 201. Themethod of claim 200, wherein Lactobacillus sp. is not detected in thesample, G. vaginalis is detected in the sample.
 202. The method of claim200, wherein G. vaginalis is detected in the sample at more than 1.4e10copies/mL.
 203. An oligomer combination for determining the presence orabsence of each of Lactobacillus sp. and G. vaginalis in a sample, theoligomer combination comprising: (a) first and secondLactobacillus-specific amplification oligomers for amplifying a targetregion of a Lactobacillus sp. target nucleic acid, wherein (i) the firstLactobacillus-specific amplification oligomer comprises a firstLactobacillus-specific target-hybridizing sequence substantiallycorresponding to the nucleotide sequence of residues 28-45 of SEQ IDNO:7 or residues 28-45 of SEQ ID NO:8 and (ii) the secondLactobacillus-specific amplification oligomer comprises a secondLactobacillus-specific target-hybridizing sequence substantiallycorresponding to the nucleotide sequence of SEQ ID NO:6; and (b) firstand second G. vaginalis-specific amplification oligomers for amplifyinga target region of a G. vaginalis target nucleic acid, wherein (i) thefirst G. vaginalis-specific amplification oligomer comprises a first G.vaginalis-specific target-hybridizing sequence substantiallycorresponding to the nucleotide sequence of residues 28-45 of SEQ IDNO:13 and (ii) the second G. vaginalis-specific amplification oligomercomprises a second G. vaginalis-specific target-hybridizing sequencesubstantially corresponding to the nucleotide sequence of SEQ ID NO:12.204. A method for determining the presence or absence of BacterialVaginosis (BV) in a subject, the method comprising: (a) providing asample from a subject suspected of having BV; (b) performing an assayfor the detection of Lactobacillus sp. and G. vaginalis in the sample;(c) for each of Lactobacillus sp. and G. vaginalis, assigning a statusof either positive or negative based on the detection assay; and (d)determining the presence or absence of BV in the subject based on acombination of the assigned Lactobacillus sp. status and G. vaginalisstatus, from step (c), wherein (i) a negative status for G. vaginalisindicates the absence of BV in the subject, (ii) if the status of G.vaginalis is positive, then a positive status for Lactobacillus sp.indicates the absence of BV in the subject and a negative status forLactobacillus sp. indicates the presence of BV in the subject.
 205. Acomposition comprising: a first detection probe that specificallyhybridizes to a Lactobacillus sp. target nucleic acid, and a seconddetection probe that specifically hybridizes to a G. vaginalis targetnucleic acid, wherein at least one of the first and second detectionprobes comprises a label, and wherein said composition (i) does notcomprise a detection probe that specifically hybridizes to a targetnucleic acid from any fungal species and (ii) does not comprise adetection probe that specifically hybridizes to a target nucleic acidfrom any bacterial species other than Lactobacillus sp. or G. vaginalis.206. A kit comprising: a first detection probe that specificallyhybridizes to a Lactobacillus sp. target nucleic acid, and a seconddetection probe that specifically hybridizes to a G. vaginalis targetnucleic acid, wherein at least one of the first and second detectionprobes comprises a label, and wherein said kit (i) does not comprise adetection probe that specifically hybridizes to a target nucleic acidfrom any fungal species and (ii) does not comprise a detection probethat specifically hybridizes to a target nucleic acid from any bacterialspecies other than Lactobacillus sp. or G. vaginalis.
 207. Thecomposition of claim 205 or the kit of claim 206, wherein the firstdetection probe specifically hybridizes to at least one of an L. gasseritarget nucleic acid, an L. crispatus target nucleic acid, and an L.jensenii target nucleic acid.
 208. The composition of claim 205 or thekit of claim 206, wherein the first detection probe targets aLactobacillus sp. 16S rRNA region corresponding to a region of SEQ IDNO:1 from about nucleotide position 91 to about nucleotide position 265,and/or the second detection probe targets a G. vaginalis 16S rRNA regioncorresponding to a region of SEQ ID NO:3 from about nucleotide position964 to about nucleotide position
 1036. 209. The composition of claim 205or the kit of claim 206, wherein the first detection probe comprises atarget-hybridizing sequence substantially corresponding to SEQ ID NO:9,and/or the second detection probe comprises a target-hybridizingsequence substantially corresponding to residues 1-19 of SEQ ID NO:14.210. The composition of claim 205 or the kit of claim 206, wherein thefirst detection probe has the nucleotide sequence of SEQ ID NO:9, and/orthe second detection probe has the nucleotide sequence of SEQ ID NO:14.211. The composition or kit of claim 209 or 210, wherein saidcomposition or kit further comprises a third detection probe thatspecifically hybridizes to the Lactobacillus sp. target nucleic acid,wherein the third detection probe comprises a target-hybridizingsequence substantially corresponding to residues 6-21 of SEQ ID NO:10.212. The composition or kit of claim 211, wherein the third detectionprobe has the nucleotide sequence of SEQ ID NO:10.